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FOXA2, a member of the forkhead family of transcription factors, plays essential roles in liver development and bile acid homeostasis. In this study, we report a 2.8 Å co-crystal structure of the FOXA2 DNA-binding domain (FOXA2-DBD) bound to a DNA duplex containing a forkhead consensus binding site (GTAAACA). FOXA2-DBD adopts the canonical winged-helix fold, with helix H3 and wing 1 regions mainly mediating the DNA recognition. Although the wing 2 region was not defined in the structure, isothermal titration calorimetry (ITC) assays suggested that this region was required for optimal DNA binding. Structure comparison with the FOXA3-DBD bound to DNA revealed more major groove contacts and less minor groove contacts in the FOXA2 structure compared to the FOXA3 structure. Structure comparison with the FOXO1-DBD bound to DNA showed that different forkhead proteins could induce different DNA conformations upon binding to identical DNA sequences. Our findings provide the structural basis for FOXA2 protein binding to a consensus forkhead site and elucidate how members of the forkhead protein family bind different DNA sites.

Vaccinia TopIB (vTopIB), a 314-amino acid eukaryal type IB topoisomerase, recognizes and transesterifies at the DNA sequence 5’-(T/C)CCTT↓, leading to the formation of a covalent DNA-(3’-phosphotyrosyl(274))-enzyme intermediate in the supercoil relaxation reaction. The C-terminal segment of vTopIB (amino acids 81-314), which engages the DNA minor groove at the scissile phosphodiester, comprises an autonomous catalytic domain that retains cleavage specificity, albeit with lower cleavage site affinity compared to the full-length enzyme. The N-terminal domain (amino acids 1-80) engages the major groove on the DNA face opposite the scissile phosphodiester. Whereas DNA contacts of the N-terminal domain have been implicated in DNA site affinity of vTopIB, it was not known whether the N-terminal domain per se could bind DNA. Here, using isothermal titration calorimetry, we demonstrate the ability of the isolated N-terminal domain to bind a CCCTT-containing 24-mer duplex with an apparent affinity that is ~2.2-fold higher than that for an otherwise identical duplex in which the pentapyrimidine sequence is changed to ACGTG. Analyses of the interactions of the isolated N-terminal domain with duplex DNA via solution NMR methods are consistent with its DNA contacts observed in DNA-bound crystal structures of full-length vTopIB. The chemical shift perturbations and changes in hydrodynamic properties triggered by CCCTT DNA versus non-CCCTT DNA suggest differences in DNA binding dynamics. The importance of key N-terminal domain contacts in the context of full-length vTopIB are underscored by assessing the effects of double-alanine mutations on DNA transesterification and its sensitivity to ionic strength.

FLI1 (Friend leukemia integration 1) is a metazoan transcription factor that is upregulated in a number of cancers. In addition, rearrangements of the fli1 gene cause sarcomas, leukemias, and lymphomas. These rearrangements encode oncogenic transcription factors, in which the DNA binding domain (DBD or ETS domain) of FLI1 on the C-terminal side is fused to a part of an another protein on the N-terminal side. Such abnormal cancer cell-specific fusions retain the DNA binding properties of FLI1 and acquire non-native protein-protein or protein-nucleic acid interactions of the substituted region. As a result, these fusions trigger oncogenic transcriptional reprogramming of the host cell. Interactions of FLI1 fusions with other proteins and with itself play a critical role in the oncogenic regulatory functions, and they are currently under intense scrutiny, mechanistically and as potential novel anticancer drug targets. We report elusive crystal structures of the FLI1 DBD, alone and in complex with cognate DNA containing a GGAA recognition sequence. Both structures reveal a previously unrecognized dimer of this domain, consistent with its dimerization in solution. The homodimerization interface is helix-swapped and dominated by hydrophobic interactions, including those between two interlocking Phe362 residues. A mutation of Phe362 to an alanine disrupted the propensity of this domain to dimerize without perturbing its structure or the DNA binding function, consistent with the structural observations. We propose that FLI1 DBD dimerization plays a role in transcriptional activation and repression by FLI1 and its fusions at promoters containing multiple FLI1 binding sites.

The formation of the pre-replicative complex (pre-RC) during the G1 phase, which is also called the licensing of DNA replication, is the initial and essential step of faithful DNA replication during the subsequent S phase. It is widely accepted that in the pre-RC, double-stranded DNA passes through the holes of two ring-shaped minichromosome maintenance (MCM) 2-7 hexamers; however, the spatial organization of the DNA and proteins involved in pre-RC formation is unclear. Here we reconstituted the pre-RC from purified DNA and proteins and visualized the complex using atomic force microscopy (AFM). AFM revealed that the MCM double hexamers formed elliptical particles on DNA. Analysis of the angle of binding of DNA to the MCM double hexamer suggests that the DNA does not completely pass through both holes of the MCM hexamers, possibly because the DNA exited from the gap between Mcm2 and Mcm5. A DNA loop fastened by the MCM double hexamer was detected in pre-RC samples reconstituted from purified proteins as well as those purified from yeast cells, suggesting a higher-order architecture of the loaded MCM hexamers and DNA strands.

Saccharomyces cerevisiae repressor-activator protein 1 (Rap1) is an essential protein involved in multiple steps of DNA regulation, as an activator in transcription, as a repressor at silencer elements, and as a major component of the shelterin-like complex at telomeres. All the known functions of Rap1 require the known high-affinity and specific interaction of the DNA-binding domain with its recognition sequences. In this work, we focus on the interaction of the DNA-binding domain of Rap1 (Rap1(DBD)) with double-stranded DNA substrates. Unexpectedly, we found that while Rap1(DBD) forms a high-affinity 1:1 complex with its DNA recognition site, it can also form lower-affinity complexes with higher stoichiometries on DNA. These lower-affinity interactions are independent of the presence of the recognition sequence, and we propose they originate from the ability of Rap1(DBD) to bind to DNA in two different binding modes. In one high-affinity binding mode, Rap1(DBD) likely binds in the conformation observed in the available crystal structures. In the other alternative lower-affinity binding mode, we propose that a single Myb-like domain of the Rap1(DBD) makes interactions with DNA, allowing for more than one protein molecule to bind to the DNA substrates. Our findings suggest that the Rap1(DBD) does not simply target the protein to its recognition sequence but rather it might be a possible point of regulation.

SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like1 (SMARCAL1) is a recently identified DNA damage response protein involved in remodeling stalled replication forks. The eukaryotic single-strand DNA binding protein replication protein A (RPA) recruits SMARCAL1 to stalled forks in vivo and facilitates regression of forks containing leading strand gaps. Both activities are mediated by a direct interaction between an RPA binding motif (RBM) at the N-terminus of SMARCAL1 and the C-terminal winged-helix domain of the RPA 32 kDa subunit (RPA32C). Here we report a biophysical and structural characterization of the SMARCAL1-RPA interaction. Isothermal titration calorimetry and circular dichroism spectroscopy revealed that RPA32C binds SMARCAL1-RBM with a Kd of 2.5 μM and induces a disorder-to-helix transition. The crystal structure of RPA32C was refined to 1.4 Å resolution, and the SMARCAL1-RBM binding site was mapped on the structure on the basis of nuclear magnetic resonance chemical shift perturbations. Conservation of the interaction surface to other RBM-containing proteins allowed construction of a model for the RPA32C/SMARCAL1-RBM complex. The implications of our results are discussed with respect to the recruitment of SMARCAL1 and other DNA damage response and repair proteins to stalled replication forks.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear protein that plays key roles in several fundamental cellular processes. PARP-1 catalyzes the polymerization of nicotinamide adenine dinucleotide on itself and other acceptor proteins, forming long branched poly(ADP-ribose) polymers. The catalytic activity of PARP-1 is stimulated upon binding to damaged DNA, but how this signal is transmitted from the N-terminal DNA binding domain to the C-terminal catalytic domain in the context of the full-length enzyme is unknown. In this paper, small-angle X-ray scattering experiments and molecular dynamics simulations were used to gain insight into the conformational changes that occur during the catalytic activation of PARP-1 by an 8-mer DNA ligand. The data are consistent with a model in which binding of the DNA ligand establishes interdomain interactions between the DNA binding and catalytic domains, which induces an allosteric change in the active site that promotes catalysis. Moreover, the PARP-1-8-mer complex is seen to adopt a conformation that is poised to recruit DNA repair factors to the site of DNA damage. This study provides the first structural information about the DNA-induced conformational activation of full-length PARP-1.

In human and mouse stem cells and brain, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) can occur outside of CG dinucleotides. Using protein binding microarrays (PBMs) containing 60-mer DNA probes, we evaluated the effect of 5mC and 5hmC on one DNA strand on the double-stranded DNA binding of the mouse B-ZIP transcription factors (TFs) CREB1, ATF1, and JUND. 5mC inhibited binding of CREB1 to the canonical CRE half-site |GTCA but enhanced binding to the C/EBP half-site |GCAA. 5hmC inhibited binding of CREB1 to all 8-mers except TGAT|GCAA, where binding is enhanced. We observed similar DNA binding patterns with ATF1, a closely related B-ZIP domain. In contrast, both 5mC and 5hmC inhibited binding of JUND. These results identify new DNA sequences that are well-bound by CREB1 and ATF1 only when they contain 5mC or 5hmC. Analysis of two X-ray structures examines the consequences of 5mC and 5hmC on DNA binding by CREB and FOS|JUN.

The transcription of many genes, particularly in prokaryotes, is controlled by transcription factors whose activity can be modulated by controlling their DNA binding affinity. Understanding the molecular mechanisms by which DNA binding affinity is regulated is important, but because forming definitive conclusions usually requires detailed structural information in combination with data from extensive biophysical, biochemical, and sometimes genetic experiments, little is truly understood about this topic. This review describes the biological requirements placed upon DNA binding transcription factors and their consequent properties, particularly the ways that DNA binding affinity can be modulated and methods for its study. What is known and not known about the mechanisms modulating the DNA binding affinity of a number of prokaryotic transcription factors, including CAP and lac repressor, is provided.

The affinity of the Ets-1 transcription factor for DNA is autoinhibited by an intrinsically disordered serine-rich region (SRR) and a helical inhibitory module (IM) appended to its winged helix–turn–helix ETS domain. Using NMR spectroscopy, we investigated how Ets-1 recognizes specific versus nonspecific DNA, with a focus on the roles of protein dynamics and autoinhibition in these processes. Upon binding either DNA, the two marginally stable N-terminal helices of the IM predominantly unfold, but still sample partially ordered conformations. Also, on the basis of amide chemical shift perturbation mapping, Ets-1 associates with both specific and nonspecific DNA through the same canonical ETS domain interface. These interactions are structurally independent of the SRR, and thus autoinhibition does not impart DNA-binding specificity. However, relative to the pronounced NMR spectroscopic changes in Ets-1 resulting from specific DNA binding, the spectra of the nonspecific DNA complexes showed conformational exchange broadening and lacked several diagnostic amide and indole signals attributable to hydrogen bonding interactions seen in reported X-ray crystallographic structures of this transcription factor with its cognate DNA sequences. Such differences are highlighted by the chemical shift and relaxation properties of several interfacial lysine and arginine side chains. Collectively, these data support a general model in which Ets-1 interacts with nonspecific DNA via dynamic electrostatic interactions, whereas hydrogen bonding drives the formation of well-ordered complexes with specific DNA.

Forkhead box (FOX) transcription factors share a conserved forkhead DNA binding domain (FHD) and are key role players in the development of many eukaryotic species. Their involvement in various congenital disorders and cancers makes them clinically relevant targets for novel therapeutic strategies. Among them, the FOXP subfamily of multidomain transcriptional repressors is unique in its ability to form DNA binding homo and heterodimers. The truncated FOXP2 FHD, in the absence of the leucine zipper, exists in equilibrium between monomeric and domain-swapped dimeric states in vitro. As a consequence, determining the DNA binding properties of the FOXP2 FHD becomes inherently difficult. In this work, two FOXP2 FHD hinge loop mutants have been generated to successfully prevent both the formation (A539P) and the dissociation (F541C) of the homodimers. This allows for the separation of the two species for downstream DNA binding studies. Comparison of DNA binding of the different species using electrophoretic mobility shift assay, fluorescence anisotropy and isothermal titration calorimetry indicates that the wild-type FOXP2 FHD binds DNA as a monomer. However, comparison of the DNA-binding energetics of the monomer and wild-type FHD, reveals that there is a difference in the mechanism of binding between the two species. We conclude that the naturally occurring reverse mutation (P539A) seen in the FOXP subfamily increases DNA binding affinity and may increase the potential for nonspecific binding compared to other FOX family members.

Dss1 is an intrinsically unstructured polypeptide that partners with the much larger Brh2 protein, the BRCA2 ortholog in Ustilago maydis, to form a tight complex. Mutants lacking Dss1 have essentially the same phenotype as mutants defective in Brh2, implying that through physical interaction Dss1 serves as a positive activator of Brh2. Dss1 associates with Brh2 through an interaction surface in the carboxy-terminal region. Certain derivatives of Brh2 lacking this interaction surface remain highly competent in DNA repair as long as a DNA-binding domain is present. However, the Dss1-independent activity raises the question of what function might be met in the native protein by having Brh2 under Dss1 control. Using a set of Brh2 fusions and truncated derivatives, we show here that Dss1 is capable of exerting control when there is a cognate Dss1-interacting surface present. We find that association of Dss1 attenuates the DNA binding potential of Brh2 and that the amino-terminal domain of Brh2 helps evict Dss1 from its carboxy-terminal interaction surface. The findings presented here add to the notion that Dss1 serves in a regulatory capacity to dictate order in association of Brh2’s amino-terminal and carboxy-terminal domains with DNA.

Differentiation of binding accurate DNA replication polymerases over error prone DNA lesion bypass polymerases is essential for the proper maintenance of the genome. The hyperthermophilic archaeal organism Sulfolobus solfataricus (Sso) contains both a B-family replication (Dpo1) and a Y-family repair (Dpo4) polymerase and serves as a model system for understanding molecular mechanisms and assemblies for DNA replication and repair protein complexes. Protein cross-linking, isothermal titration calorimetry, and analytical ultracentrifugation have confirmed a previously unrecognized dimeric Dpo4 complex bound to DNA. Binding discrimination between these polymerases on model DNA templates is complicated by the fact that multiple oligomeric species are influenced by concentration and temperature. Temperature-dependent fluorescence anisotropy equilibrium binding experiments were used to separate discrete binding events for the formation of trimeric Dpo1 and dimeric Dpo4 complexes on DNA. The associated equilibria are found to be temperature-dependent, generally leading to improved binding at higher temperatures for both polymerases. At high temperatures, DNA binding of Dpo1 monomer is favored over binding of Dpo4 monomer, but binding of Dpo1 trimer is even more strongly favored over binding of Dpo4 dimer, thus providing thermodynamic selection. Greater processivities of nucleotide incorporation for trimeric Dpo1 and dimeric Dpo4 are also observed at higher temperatures, providing biochemical validation for the influence of tightly bound oligomeric polymerases. These results separate, quantify, and confirm individual and sequential processes leading to the formation of oligomeric Dpo1 and Dpo4 assemblies on DNA and provide for a concentration- and temperature-dependent discrimination of binding undamaged DNA templates at physiological temperatures.

Rev1 is a translesion synthesis (TLS) DNA polymerase essential for DNA damage tolerance in eukaryotes. In the process of TLS stalled high-fidelity replicative DNA polymerases are temporarily replaced by specialized TLS enzymes that can bypass sites of DNA damage (lesions), thus allowing replication to continue or postreplicational gaps to be filled. Despite its limited catalytic activity, human Rev1 plays a key role in TLS by serving as a scaffold that provides an access of Y-family TLS polymerases polη, ι, and κ to their cognate DNA lesions and facilitates their subsequent exchange to polζ that extends the distorted DNA primer-template. Rev1 interaction with the other major human TLS polymerases, polη, ι, κ, and the regulatory subunit Rev7 of polζ, is mediated by Rev1 C-terminal domain (Rev1-CT). We used NMR spectroscopy to determine the spatial structure of the Rev1-CT domain (residues 1157-1251) and its complex with Rev1 interacting region (RIR) from polη (residues 524-539). The domain forms a four-helix bundle with a well-structured N-terminal β-hairpin docking against helices 1 and 2, creating a binding pocket for the two conserved Phe residues of the RIR motif that upon binding folds into an α-helix. NMR spin-relaxation and NMR relaxation dispersion measurements suggest that free Rev1-CT and Rev1-CT/polη-RIR complex exhibit μs-ms conformational dynamics encompassing the RIR binding site, which might facilitate selection of the molecular configuration optimal for binding. These results offer new insights into the control of TLS in human cells by providing a structural basis for understanding the recognition of the Rev1-CT by Y-family DNA polymerases.

The Saccharomyces cerevisiae high mobility group protein HMO2 is a component of the chromatin remodeling complex INO80. In this capacity, it has been shown to direct INO80 to DNA double-strand breaks, thereby contributing to double-strand break repair. Consistent with such function, HMO2 binds DNA ends, protecting them from exonucleolytic degradation. We show here that both domains of HMO2, HMO2-BoxA and HMO2-BoxB, bind preferentially to distorted DNA, with HMO2-BoxA binding preferentially to four-way DNA junctions and DNA with tandem mismatches and HMO2-BoxB binding four-way junctions as well as DNA with stem-loop structures, tandem mismatches, and abasic sites. As previously reported for mammalian high mobility group proteins, the acidic C-terminal extension significantly attenuates DNA binding. Notably, the unique ability of HMO2 to protect DNA ends is conferred by the Box A domain. Considering the reported roles for INO80 in other events such as recovery of stalled replication forks and nucleotide excision repair, we assessed the effect of DNA damaging agents on an hmo2Δ strain; while modest growth inhibition is seen upon exposure to UV light, exposure to hydroxyurea, which causes replication fork arrest, induces severe growth deficiency. These data suggest that HMO2 may also participate in directing the INO80 complex to sites such as stalled replication forks; the preferred binding of HMO2 domains to damaged DNA and intermediates in homologous recombination is consistent with such function.

DNA-based nanomechanical devices can be used to characterize the action of DNA-distorting proteins. Here, we have constructed a device wherein two DNA triple crossover (TX) molecules are connected by a shaft, similar to a previous device that measured the binding free energy of integration host factor. In the present case, the binding site on the shaft contains the sequence recognized by SoxR protein, the apo-form of which is a transcriptional activator. Another active form is oxidized [2Fe-2S] SoxR formed during redox sensing, and previous data suggest that activated Fe-SoxR distorts its binding site by localized DNA untwisting by an amount that corresponds to about two base pairs. A pair of dyes report the fluorescence resonance energy transfer (FRET) signal between the two TX domains, reflecting changes in the shape of the device upon binding the protein. The TX domains are used to amplify the signal expected from a relatively small distortion of the DNA binding site. From FRET analysis of apo-SoxR binding, the effect of apo-SoxR on the original TX device is similar to the effect of shortening the TX device by 2 bp. We estimate that the binding free energy of apo-SoxR on the DNA target site is 3.2-6.1 kcal/mol.

To efficiently repair DNA, human alkyladenine DNA glycosylase (AAG) must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process and a higher-affinity interaction for catalytic repair. Here, we present crystal structures of AAG trapped in two DNA-bound states. The lower-affinity depiction allows us to investigate, for the first time, the conformation of this protein in the absence of a tightly bound DNA adduct. We find that active site residues of AAG involved in binding lesion bases are in a disordered state. Furthermore, two loops that contribute significantly to the positive electrostatic surface of AAG are disordered. Additionally, a higher-affinity state of AAG captured here provides a fortuitous snapshot of how this enzyme interacts with a DNA adduct that resembles a one-base loop.

HMGB1, one of the most abundant nuclear proteins, has a strong binding affinity for cisplatin-modified DNA. It has been proposed that HMGB1 enhances the anticancer efficacy of cisplatin by shielding platinated DNA lesions from repair. Two cysteine residues in HMGB1 domain A form a reversible disulfide bond under mildly oxidizing conditions. The reduced domain A protein binds to a 25-bp DNA probe containing a central 1,2-d(GpG) intrastrand cross-link, the major platinum-DNA adduct, with a 10-fold greater binding affinity than the oxidized domain A. The binding affinities of singly and doubly mutated HMGB1 domain A, respectively deficient in one or both cysteine residues that form the disulfide bond, are unaffected by changes in external redox conditions. The redox-dependent nature of the binding of HMGB1 domain A to cisplatin-modified DNA suggests that formation of the intradomain disulfide bond induces a conformational change that disfavors binding to cisplatin-modified DNA. Hydroxyl radical footprinting analyses of wild-type domain A bound to platinated DNA under different redox conditions revealed identical cleavage patterns, implying that the asymmetric binding mode of the protein across from the platinated lesion is conserved irrespective of the redox state. The results of this study reveal that the cellular redox environment can influence the interaction of HMGB1 with the platinated DNA and suggest that the redox state of the A domain is a potential factor in regulating the role of the protein in modulating the activity of cisplatin as an anticancer drug.

The epigenetic control of transcription requires the selective recognition of methylated CpG dinucleotides by methylation-sensitive sequence-specific DNA binding proteins. In order to probe the mechanism of selective interaction of the methyl-binding protein with methylated DNA, we have prepared a series of oligonucleotides containing modified purines and pyrimidines at the recognition site, and we have examined the binding of these oligonucleotides to the methyl-binding domain (MBD) of the methyl-CpG-binding protein 2 (MeCP2). Our results suggest that pyrimidine 5-substituents similar in size to a methyl group facilitate protein binding; however, binding affinity does not correlate with the hydrophobicity of the substituent, and neither the 4-amino group of 5-methylcytosine (mC) nor Watson-Crick base pair geometry is essential for MBD binding. However, 5-substituted uracil analogues in one strand do not direct human DNA methyltransferase 1 (DNMT1) methylation of the opposing strand, as does mC. Important recognition elements do include the guanine O6 and N7 atoms present in the major groove. Unexpectedly, removal of the guanine 2-amino group from the minor groove substantially enhances MBD binding, likely resulting from DNA bending at the substitution site. The enhanced binding of the MBD to oligonucleotides containing several cytosine analogues observed here is better explained by a DNA-protein interface mediated by structured water as opposed to hydrophobic interactions.

Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a "jackknife model" in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.

The mammalian high-mobility group protein AT hook 2 (HMGA2) is a DNA binding protein that specifically recognizes the minor groove of AT-rich DNA sequences. Disruption of its expression pattern is directly linked to oncogenesis and obesity. In this paper, we constructed a new plasmid pBendAT to study HMGA2-induced DNA bending. pBendAT carries a 230 bp DNA segment containing five pairs of restriction enzyme sites, which can be used to produce a set of DNA fragments of identical length to study protein-induced DNA bending. The DNA fragments of identical length can also be generated using PCR amplification. Since pBendAT does not contain more than three consecutive AT base pairs, it is suitable for the assessment of DNA bending induced by proteins recognizing AT-rich DNA sequences. Indeed, using pBendAT, we demonstrated that HMGA2 is a DNA bending protein and bends all three tested DNA binding sequences of HMGA2, SELEX1, SELEX2, and PRDII. The DNA bending angles were estimated to be 34.2 degrees , 33.5 degrees , and 35.4 degrees , respectively.

Brh2, the BRCA2 homologue in Ustilago maydis, plays a crucial role in homologous recombination by controlling Rad51. In turn, Brh2 is governed by Dss1, an intrinsically disordered protein that forms a tight complex with the C-terminal region of Brh2. This region of the protein associating with Dss1 is highly conserved in sequence and by comparison with mammalian BRCA2 corresponds to a part of the DNA binding domain with characteristic OB folds. The N-terminal region of Brh2 harbors a less-defined but powerful DNA binding site, the activity of which is revealed upon deletion of the C-terminal region. Full-length Brh2 complexed with Dss1 binds DNA slowly, while the N-terminal fragment binds quickly. The DNA binding activity of full-length Brh2 appears to correlate with dissociation of Dss1. Addition of Dss1 to the heterotypic Brh2-Dss1 complex attenuates DNA binding activity, but not by direct competition for the N-terminal DNA binding site. Conversely, the Brh2-Dss1 complex dissociates more quickly when DNA is present. These findings suggest a model in which binding of Brh2 to DNA is subject to allosteric regulation by Dss1.

Enzymatic sequence-specific DNA modification involves multiple poorly understood intermediates. DNA methyltransferases like M.HhaI initially bind nonspecific DNA and then selectively bind and modify a unique sequence. High-resolution NMR was used to map conformational changes occurring in M.HhaI upon binding nonspecific DNA, a one base pair altered noncognate DNA sequence, and both hemimethylated and unmethylated cognate DNA sequences. Comparisons with previous NMR studies of the apoenzyme and enzyme-cofactor complex provide snapshots of the pathway to sequence-specific complex formation. Dramatic chemical shift perturbations reaching many distal sites within the protein are detected with cognate DNA, while much smaller changes are observed upon nonspecific and noncognate DNA binding. A cooperative rather than stepwise transition from a nonspecific to a cognate complex is revealed. Furthermore, switching from unmethylated to hemimethylated cognate DNA involves detectable protein conformational changes 20-30 A away from the methyl group, indicating high protein sensitivity and plasticity to DNA modification.

HMGB proteins are abundant non-histone components of eukaryotic chromatin. The biological function of DNA sequence-nonspecific HMGB proteins is obscure. These proteins are composed of one or two conserved HMG box domains, each forming three alpha-helices that fold into a sequence-nonspecific DNA-binding module recognizing the DNA minor groove. Box A and box B homology domains have subtle sequence differences such that box B domains bend DNA strongly while DNA bending by isolated box A domains is weaker. Both box A and box B domains preferentially bind to distorted DNA structures. Here we show using DNA cyclization kinetics assays in vitro and Escherichia coli DNA looping assays in vivo that an isolated HMG box A domain derived from human HMGB2 folds poorly and does not enhance apparent DNA flexibility. Surprisingly, substitution of a small number of cationic residues from the N-terminal leader of a functional yeast box B protein, Nhp6Ap, confers the ability to enhance DNA flexibility. These results demonstrate important roles for cationic leader amino acids in HMGB folding, DNA interaction, and DNA bending.

DNA polymerases and aminoacyl-tRNA synthetases (ARSs) represent large enzyme families with critical roles in the transformation of genetic information from DNA to RNA to protein. DNA polymerases carry out replication and collaborate in the repair of the genome, while ARSs provide aminoacylated tRNA precursors for protein synthesis. Enzymes of both families face the common challenge of selecting their cognate small molecule substrates from a pool of chemically related molecules, achieving high levels of discrimination with the assistance of proofreading mechanisms. Here, the fidelity preservation mechanisms in these two important systems are reviewed, and similar features highlighted. Among the noteworthy features common to both DNA polymerases and ARSs are the use of multidomain architectures that segregate synthetic and proofreading functions into discrete domains; the use of induced fit to enhance binding selectivity; the imposition of fidelity at the level of chemistry; and the use of post-chemistry error correction mechanisms to hydrolyze incorrect products in a discrete editing domain. These latter mechanisms further share the common property that error correction involves the translocation of mis-incorporated products from the synthetic to the editing site, and that the accuracy of the process may be influenced by the rates of translocation in either direction. Fidelity control in both families can thus be said to rely on multiple elementary steps, each with its contribution to overall fidelity. The summed contribution of these “kinetic checkpoints” provides the high observed overall accuracy of DNA replication and aminoacylation.

Alternate DNA structures other than double-stranded B-form DNA can potentially impede cellular processes such as transcription and replication. The DNA triplex helix and G4 tetraplex structures that form by Hoogsteen hydrogen bonding are two examples of alternate DNA structures that can be a source of genomic instability. In this study, we have examined the ability of human replication protein A (RPA), a single-stranded DNA binding protein that is implicated in all facets of DNA metabolism, to destabilize DNA triplexes and tetraplexes. Biochemical studies demonstrate that RPA efficiently melts an intermolecular DNA triple helix consisting of a pyrimidine motif third strand annealed to a 4 kb duplex DNA fragment at protein concentrations equimolar to the triplex substrate. Heterologous single-stranded DNA binding proteins ( Escherichia coli SSB, T4 gene 32) melt the triplex substrate very poorly or not at all, suggesting that the triplex destabilizing effect of RPA is specific. In contrast to the robust activity on DNA triplexes, RPA does not melt intermolecular G4 tetraplex structures. Cellular assays demonstrated increased triplex DNA content when RPA is transiently repressed, suggesting that RPA melting of triple helical structures is physiologically important. On the basis of our results, we suggest that the abundance of RPA known to exist in vivo is likely to be a strong deterrent to the stability of triplexes that can potentially form from human genomic DNA sequences.

The methyl-CpG binding domain (MBD) is a conserved domain in transcriptional factors that binds to methylated CpG dinucleotide DNA sequences in vertebrates. The complex is comprised of an asymmetric MBD monomer and a symmetric DNA duplex. Therefore, in the complex, each strand of the duplex DNA is in contact with the protein at a distinct surface and thus exhibits a different chemical shift in NMR spectra. Two-dimensional chemical exchange spectroscopy revealed the presence of a stochastic exchange of the two strands of the duplex DNA in the complex at a rate of 4 s (-1) at 25 degrees C, which indicates the existence of a motion of the MBD such that the orientation of the MBD becomes reversed with respect to the DNA duplex. Kinetic and thermodynamic analyses using surface plasmon resonance, quartz crystal microbalance, and isothermal titration calorimetry suggest that the reversal of MBD with respect to the DNA duplex takes place without its complete dissociation from DNA, indicating the presence of an intermediate protein-DNA binding state that allows the protein to undergo a flip motion upon DNA.

Human telomeric repeat binding factor 2 (hTRF2) is a protein that plays an important role in capping human telomeres to protect them from DNA damage repair systems. The ineffectiveness of hTRF2 may be linked to aging and cancer. We report the use of PLIMSTEX (protein-ligand interactions by mass spectrometry, titration, and H/D exchange) and selective acetylation of lysine residues to study the interaction of the DNA-binding domain and double-stranded telomeric DNA (repeats of TTAGGG). By increasing the resolution of PLIMSTEX to the peptide level, we localized the changes in deuterium uptake of hTRF2 as a function of varying amounts of a model oligodeoxynucleotide. From these experiments, we determined the affinity constant for binding to DNA, which is within a factor of 3 of the previously reported value. Amide H/D exchange revealed portions of the protein that have contacts with the phosphate backbone of DNA, whereas acetylation disclosed the decrease in solvent accessibility of regions containing Lys 447 and 488, which must be involved in interactions with the DNA major and minor grooves. These complementary approaches of amide H/D exchange and selective side chain modification can be employed effectively to pinpoint and quantify protein-ligand, in particular protein-DNA, interactions.

Bacteriophage lambda integrase (lambda-Int), a phage-encoded DNA recombinase, cleaves its substrate DNA to facilitate the formation and later resolution of a Holliday junction intermediate during recombination. The core-binding and catalytic domains of lambda-Int constitute a bipartite enzyme that mediates site-specific DNA cleavage through their interactions with opposite sides of the recognition sequence. Despite minimal direct contact between the domains, the core-binding domain has been shown to facilitate site-specific DNA cleavage when provided in trans, indicating that it plays a role beyond enhancing binding affinity. Biophysical characterization of the core-binding domain and its interactions with DNA reveal that the domain is poorly structured in its free form and folds upon binding to DNA. Folding of the protein is accompanied by induced-fit structural changes in the DNA ligand. These data support a model by which the core-binding domain plays a catalytic role by reshaping the substrate DNA for effective cleavage by the catalytic domain.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a transcriptional factor involved in cell differentiation and transformation. Disruption of its normal expression pattern is directly linked to oncogenesis and obesity. HMGA2 contains three "AT-hook" DNA binding domains, which specifically bind to the minor groove of AT-rich sequences. Using a PCR-based systematic evolution of ligands by exponential enrichment (SELEX) procedure, we have identified two consensus sequences for HMGA2, 5'-ATATTCGCGAWWATT-3' and 5'-ATATTGCGCAWWATT-3', where W represents A or T. These two consensus sequences have a unique and interesting feature: the first five base pairs are AT-rich, the middle four base pairs are GC-rich, and the last six base pairs are AT-rich. Our results showed that all three of these segments are critical for high-affinity binding of HMGA2 to DNA. For example, if one of the AT-rich sequences is mutated to a non-AT-rich sequence, the DNA binding affinity of HMGA2 is reduced at least 100-fold. Intriguingly, if the GC-segment is replaced by an AT-rich segment, the binding affinity of HMGA2 is reduced approximately 5-fold. Identification of the consensus sequences for HMGA2 represents an important step toward finding its binding sites within the genome.

Estrogen receptor alpha (ER) is a member of the nuclear hormone receptor family, which upon binding estrogen shows increased apparent affinity for nuclear components (tight nuclear binding). The nuclear components that mediate this tight nuclear binding have been proposed to include both ER-DNA interactions and ER-protein interactions. In this paper, we demonstrate that tight nuclear binding of ER upon estrogen occupation requires ER-DNA interactions. Hormone-bound ER can be extracted from the nucleus in low-salt buffer using various polyanions, which mimic the phosphate backbone of DNA. The importance of specific ER-DNA interactions in mediating tight nuclear binding is also supported by the 380-fold lower concentration of the ERE oligonucleotide necessary to extract estrogen-occupied ER from the nucleus compared to the polyanions. We also demonstrate that estrogen-induced tight nuclear binding requires both the nuclear localization domain and the DNA binding domain of ER. Finally, enzymatic degradation of nuclear DNA allows us to recover 45% of tight nuclear-bound ER. We further demonstrate that ER-AIB1 interaction is not required for estrogen-induced tight nuclear binding. Taken together, we propose a model in which tight nuclear binding of the estrogen-occupied ER is predominantly mediated by ER-DNA interactions. The effects of estrogen binding on altering DNA binding in whole cells are proposed to occur through estrogen-induced changes in ER-chaperone protein interactions, which alter the DNA accessibility of ER but do not directly change the affinity of the ER for DNA, which is similar for both unoccupied and occupied ER.

We have studied the tetramer-dimer equilibrium of histones H3/H4 and its effect on DNA supercoiling. Two approaches were found to shift the equilibrium toward dimer. In both instances, when deposited on DNA, the dimers formed positively coiled DNA. The first approach was to modify cysteine 110 of H3 with 5,5'-dithio-bis(2-nitrobenzoic acid (DTNB) and to directly add the histones to DNA at physiological ionic strength. The second approach involved adding an excess of the histone chaperone, nucleosome assembly protein 1 (NAP1) to the H3/H4 prior to deposition on the DNA. It was also observed that when H3/H4 were deposited in the tetrameric state, negatively coiled DNA was formed. The topological state of the DNA prior to deposition was also found to influence the final conformational state of H3/H4. It is proposed that in the tetrameric state, the H3-H3 interface has a left-handed pitch prior to binding DNA. In the dimeric state, the H3-H3 interface is not established until bound to DNA, at which point either the left or right-handed pitch will form on the basis of the initial topology of the DNA. Formaldehyde cross-linking and reversal were applied to identify the histone-histone interactions that facilitate the formation of positive stress. Higher-order interactions between multiple H3/H4 dimers were required to propagate this specific conformation. Changes in the conformational state of H3/H4 were also observed when the histones were bound to DNA prior to treatment with NAP1. It is proposed that these conformational changes in H3/H4 are involved in promoter activation and transcription elongation through nucleosomes.

T7 RNA polymerase is known to induce bending of its promoter DNA upon binding, as evidenced by gel-shift assays and by recent end-to-end fluorescence energy transfer distance measurements. Crystal structures of promoter-bound and initially transcribing complexes, however, lack downstream DNA, providing no information on the overall path of the DNA through the protein. Crystal structures of the elongation complex do include downstream DNA and provide valuable guidance in the design of models for the complete melted bubble structure at initiation. In the current study, we test a specific structural model for the initiation complex, obtained by alignment of the C-terminal regions of the protein structures from both initiation and elongation and then simple transferal of the downstream DNA from the elongation complex onto the initiation complex. Fluorescence resonance energy transfer measurement of distances from a point upstream on the promoter DNA to various points along the downstream helix reproduce the expected helical periodicity in the distances and support the model's orientation and phasing of the downstream DNA. The model also makes predictions about the extent of melting downstream of the active site. By monitoring fluorescent base analogues incorporated at various positions in the DNA, we have mapped the downstream edge of the bubble, confirming the model. The initially melted bubble, in the absence of substrate, encompasses 7-8 bases and is sufficient to allow synthesis of a three base transcript before further melting is required. The results demonstrate that despite massive changes in the N-terminal portion of the protein and in the DNA upstream of the active site, the DNA downstream of the active site is virtually identical in both initiation and elongation complexes.

HMG box 5 of human upstream binding factor (hUBF) consists of three alpha-helices arranged in an L-shape with a hydrophobic core embraced by these helices and stabilized by extensive hydrophobic interactions between nonpolar residues around the core. The GdmCl-induced equilibrium unfolding transition of HMG box 5 of hUBF was monitored by both circular dichroism (CD) and fluorescence spectra. A cooperative two-state unfolding process was observed. The unfolding free energy, DeltaGU(D2O), and the cooperativity of the unfolding reaction, m, are 4.6 +/- 0.16 kcal x mol-1 and 1.62 +/- 0.06 kcal x mol-1 x M-1, respectively. Native-state hydrogen exchange (NHX) experiments under EX2 conditions were performed. NHX results clearly show that the hydrophobic core among the three helices is a slow-exchange core. The three helices would not contribute equally to the stability of the native protein. Helix 3 appears to contribute the least to the stability. The NHX data have also allowed the local, subglobal, and global unfolding structures of hUBF HMG box 5 to be dissected, and common global and subglobal unfolding units were successfully detected.

Functions of transcription factors require formation of specific complexes at particular sites in cis-regulatory elements of genes. However, chromosomal DNA contains numerous sites that are similar to the target sequences recognized by transcription factors. The influence of such “quasi-specific” sites on functions of the transcription factors is not well understood at present by experimental means. In this work, using fluorescence methods, we have investigated the influence of quasi-specific DNA sites on the efficiency of target location by the zinc finger DNA-binding domain of the inducible transcription factor Egr-1, which recognizes a 9 bp sequence. By stopped-flow assays, we measured the kinetics of Egr-1’s association with a target site on 143 bp DNA in the presence of various competitor DNAs, including nonspecific and quasi-specific sites. The presence of quasi-specific sites on competitor DNA significantly decelerated the target association by the Egr-1 protein. The impact of the quasi-specific sites depended strongly on their affinity, their concentration, and the degree of their binding to the protein. To quantitatively describe the kinetic impact of the quasi-specific sites, we derived an analytical form of the apparent kinetic rate constant for the target association and used it for fitting to the experimental data. Our kinetic data with calf thymus DNA as a competitor suggested that there are millions of high-affinity quasi-specific sites for Egr-1 among the 3 billion bp of genomic DNA. This study quantitatively demonstrates that naturally abundant quasi-specific sites on DNA can considerably impede the target search processes of sequence-specific DNA-binding proteins.

Mammalian global genomic nucleotide excision repair requires lesion recognition by XPC, whose detailed binding mechanism remains to be elucidated. Here we have delineated the dynamic molecular pathway and energetics of lesion-specific and productive binding by the Rad4/yeast XPC lesion recognition factor, as it forms the open complex [Min, J. H., and Pavletich, N. P. (2007) Nature 449, 570–575; Chen, X., et al. (2015) Nat. Commun. 6, 5849] that is required for excision. We investigated extensively a cis-syn cyclobutane pyrimidine dimer in mismatched duplex DNA, using high-level computational approaches. Our results delineate a preferred correlated motion mechanism, which provides for the first time an atomistic description of the sequence of events as Rad4 productively binds to the damaged DNA.

XPC is a 940-residue multidomain protein critical for the sensing of aberrant DNA and initiation of global genome nucleotide excision repair. The C-terminal portion of XPC (residues 492-940; XPC-C) has critical interactions with DNA, RAD23B, CETN2, and TFIIH, whereas functional roles have not yet been assigned to the N-terminal portion (residues 1-491; XPC-N). In order to analyze the molecular basis for XPC function and mutational defects associated with xeroderma pigmentosum (XP) disease, a series of stable bacterially expressed N- and C-terminal fragments were designed on the basis of sequence analysis and produced for biochemical characterization. Limited proteolysis experiments combined with mass spectrometry revealed that the full XPC-C is stable but XPC-N is not. However, a previously unrecognized folded helical structural domain was found within XPC-N, XPC(156-325). Pull-down and protease protection assays demonstrated that XPC(156-325) physically interacts with the DNA repair factor XPA, establishing the first functional role for XPC-N. XPC-C exhibits binding characteristics of the full-length protein, including stimulation of DNA binding by physical interaction with RAD23B and CETN2. Analysis of an XPC missense mutation (Trp690Ser) found in certain patients with XP disease revealed that this mutation is associated with a diminished ability to bind DNA. Evidence of contributions to protein interactions from regions in both XPC-N and XPC-C along with recently recognized homologies to yeast PNGase prompted construction of a structural model of a folded XPC core. This model offers key insights into how domains from the two portions of the protein may cooperate in generating specific XPC functions.

Mdm2 negatively regulates p53 by inhibiting its transcriptional activity and promoting its degradation by functioning as an E3 ubiquitin ligase. The primary p53 binding site on mdm2 is located in its N-terminal domain. Through binding to p53 at its N-terminal transactivation domain, mdm2 directly blocks the transcriptional activation function of p53. We discovered that truncated mdm2 protein constructs without the N-terminal p53 binding domain are at least as active as full-length mdm2 in catalyzing p53 ubiquitination. Furthermore, the deletion of the central acidic domain significantly reduces the E3 ligase activity of mdm2 toward p53. We have also performed GST pull-down experiments to probe the direct binding of various mdm2 domain constructs toward full length p53 and found that mdm2 constructs without the N-terminal p53 binding domain retain the ability to bind to p53. Our kinetic and binding data localize the second p53 binding site between amino acids 211 and 361, including the acidic domain and the zinc finger region. Our work, consistent with other reports, suggests that the p53 tetramer interacts with at least two sites on mdm2. Although the interaction between the N-termini of mdm2 and p53 blocks the transactivation activity of p53, the interaction between the central domain of mdm2 and the core domain of p53 is critical for the ubiquitination and degradation of p53. This second mdm2-p53 interaction site represents an alternative target for small molecule modulators of the mdm2-p53 pathway.

The dynamic interaction of transcription factors with DNA regulates gene expression, conferring temporal and cell-specific control in response to a wide range of intracellular and extracellular cues. However, these protein:DNA interactions are not always easily predicted, as the exact sequence of a binding site can alter its function in unexpected ways. For instance, particular sequences may influence the recruitment of co-factors by altering the conformation of the bound transcription factor via specific interactions with individual nucleotides (1-3). If a site binds transcription factors as a dimer, both spacing and orientation of the two half sites affect how the site behaves (4,5). Also, not all functional binding sites are a good match to the consensus sequence compiled from known binding sites for a particular factor (6). In addition, the interactions between transcription factors and DNA do not necessarily rely solely on the sequence of the actual binding site, but may be influenced by other DNA elements. Sequences flanking the binding site can affect response element utilization by altering the protein conformation of a factor bound to the DNA (7). Nearby sequences may bind transcription factors of their own that change the functionality of an unrelated site (8-10). Distal DNA sequences can silence transcription from a known site in a cell-specific manner (11). A more complex example is a factor:DNA interaction that recruits a corepressor to affect a transcription factor bound at a distal site (12). Location of the binding site relative to basal regulatory elements is sometimes important, as demonstrated by the positional dependence of a hormone response element in relation to the TATA box of a gene promoter (13). A single transcription factor binding site is also capable of affecting multiple genes simultaneously, sometimes over a considerable distance (14). Here, we examine interplay between two distinct determinants, flanking sequence and dimerization partner, that influence the binding and function of the glucocorticoid receptor (GR)1. The Xenopus γ-fibrinogen gene upstream regulatory region contains a binding site for a heterodimer of Xenopus glucocorticoid receptor accessory factor (XGRAF) and GR (8), which is essential for maximal hormone induction (15-17). Individually, XGRAF and GR interact with DNA to form single distinct bands in a gel mobility shift assay. Both XGRAF and GR binding are highly specific for their respective sites as confirmed by competition experiments using mutated DNA competitors (8,15,16). The combined XGRAF:GR heterodimer binding site is readily converted to a GR homodimer binding site by a single point mutation (15). Taking advantage of this property here, we used transfections and quantitative gel mobility shift assays to determine the effects of mutating individual flanking sequences on the abilities of XGRAF:GR and GR:GR to stimulate transcription.

Proteins frequently contain unstructured regions apart from a functionally important and well-conserved structured domain. Functional and structural aspects for these regions are frequently less clear. The general human positive cofactor 4 (PC4), has such a domain organization and can interact with various DNA substrates, transcriptional activators, and basal transcription factors. While essential for the cofactor function, structural and functional knowledge about these interactions is limited. Using biochemical, nuclear magnetic resonance (NMR), and docking experiments, we show that the carboxy-terminal structured core domain (PC4ctd) is required and sufficient for binding to single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and the herpes simplex virion protein 16 (VP16) activation domain (VP16ad). We determined the interaction surfaces within PC4 and showed that VP16 and DNA binding are mutually exclusive. Although the amino-terminal domain of PC4 (PC4ntd) alone is devoid of any bioactivity, it increases the interaction with VP16ad. While it decreases the ssDNA-binding and DNA-unwinding activity, it does not influence dsDNA binding. Structural characterization of this domain showed that it is highly flexible and mostly unstructured both in the free form and in the complex. NMR titration experiments using various protein and DNA substrates of the individual domains and the full-length PC4 revealed local conformational or environmental changes in both the structured and unstructured subdomains, which are interpreted to be caused by inter- and intramolecular interactions. We propose that the unstructured PC4ntd regulates the PC4 cofactor function by specific interactions with the activator and through modulation and/or shielding of the interaction surface in the structured core of PC4ctd.

Transcription of the ribosomal RNA genes of mammals by RNA polymerase I is rapidly activated by epidermal growth factor via the MAP-kinase (ERK) signaling cascade. This activation is mediated by direct phosphorylation of the HMG box DNA binding domains of the architectural transcription factor UBF. Mutation of the ERK sites of UBF inhibits its normal function and blocks growth factor activation of ribosomal transcription. UBF has little or no DNA sequence selectivity and binds throughout the ribosomal genes, defining a specialized chromatin. Indeed, the HMG boxes of UBF induce looping of the ribosomal DNA to create the enhancesome, a structure somewhat reminiscent of the nucleosome. Here, we show that both ERK phosphorylation and mutations that simulate this phosphorylation decrease the affinity of the individual HMG boxes of UBF for linear ribosomal DNA but have little or no effect on the capacity of these HMG boxes to bind to pre-bent DNA and do not affect the overall binding constant of UBF for the DNA. Electron spectroscopic imaging showed that ERK site UBF mutants do not induce the characteristic DNA looping of the enhancesome and associate with no more than half of the enhancesomal DNA. The data demonstrate that ERK phosphorylation of UBF prevents DNA bending by its first two HMG boxes, leading to a cooperative unfolding of the enhancesome.

Nucleotide excision repair (NER)1 is a versatile DNA repair pathway that exists to remove helix-distorting DNA lesions that would otherwise interfere with essential DNA-dependent processes, such as DNA replication and transcription. Together, more than 30 eukaryotic proteins are involved in four distinct steps that include initial damage recognition, cleavage of the damaged strand, displacement of the strand, and resynthesis and ligation, and the entire process has been reconstituted using purified proteins (1, 2). The initial step of DNA damage recognition can occur by either of two distinct mechanisms. The first couples NER to transcription, ensuring that the transcribed strand of genes is efficiently repaired (3). The second targets genomic DNA and achieves damage recognition through proteins that have a higher affinity for damaged DNA than for undamaged DNA. To date, several proteins have shown damage-specific binding, including the XPC–hHR23B complex (XPC–hHR23B), replication protein A (RPA), XPA, and DDB (4). Of these, only XPC–hHR23B is dispensable for transcription-coupled repair (TCR) in the presence of a stalled RNA polymerase II (5) but is specifically required for global genomic DNA repair (GGR) (6). The 144 kDa XPC protein is found in a heterotrimeric complex with hHR23B, the 44 kDa human homologue of the yeast protein Rad23 (7) and centrin 2, an 18 kDa centrosome component (8). However, XPC–hHR23B is sufficient for in vitro reconstitution of NER (9–11). Abundant evidence suggests that XPC–hHR23B is the first complex to recognize and bind to damaged DNA during GGR. For example, XPC–hHR23B is required for the earliest detectable open complex formation around a lesion at the beginning of NER (12, 13) and is the only NER protein with sufficient specificity and affinity to produce a distinct DNase footprint on DNA (14). A recent study used highly purified NER proteins and permanganate footprinting to confirm that XPC–hHR23B is the first complex to recognize damaged DNA and initiate the opening of nine bases of the helix around a site of damage (15). Although XPC–hHR23B is likely the initiator of GGR, the role of XPC in damage recognition has not been without controversy. Previous reports of the binding activity of XPC note that although XPC binds to a wide variety of lesions such as UV-induced photoproducts, cisplatin adducts, and AAF adducts, XPC also binds with high affinity to undamaged DNA (16). XPC–hHR23B is additionally able to bind with equal affinity to substrates containing bubbles with or without damage, although only the damaged substrates are able to be repaired (17). However, the preference of XPC–hHR23B for damaged versus undamaged DNA has been shown to increase up to 400-fold with the addition of undamaged competitor DNA (10). Notably, the large preference observed by XPC–hHR23B for damaged DNA in equilibrium binding experiments is consistent with either an increased rate of association or a decreased rate of dissociation from damaged DNA; however, these studies are unable to distinguish between these possibilities. In this report, we have examined the pre-steady state kinetics of binding of XPC–hHR23B to undamaged and damaged DNA using the intrinsic fluorescence of XPC–hHR23B and stopped-flow analysis to better understand the mechanism of damage-specific DNA binding. Equilibrium binding experiments were performed with the same DNA substrates to provide additional insight into the binding of XPC–hHR23B to damaged and undamaged DNA. The results revealed that damage-specific binding by XPC–hHR23B was mostly attributable to a dramatically increased kon for cisplatin and UV-damaged duplex DNA substrates compared to that for the undamaged duplex substrate, with very little difference in the dissociation rate. XPC–hHR23B displayed a 3-fold higher affinity for a 1,3 d(GpXpG) cisplatin adduct than for a 1,2 d(GpG) adduct, which was due to an increased rate of dissociation from the 1,2 d(GpG) adduct. Notably, although XPC–hHR23B binds with high affinity to single-stranded DNA and damaged duplex DNA, the presence of cisplatin damage on single-stranded DNA resulted in a decrease in the kon and an increase in the koff compared to those of undamaged single-stranded DNA, suggesting that XPC–hHR23B likely binds to the undamaged strand or adjacent to the adduct but not to the adduct itself. These data provide additional evidence that recognition and binding by XPC–hHR23B are predominantly a function of altered DNA structure.

Template Activating Factor (TAF) I remodels the adenovirus (Ad) core structure composed of the Ad genome DNA and basic viral core proteins and stimulates in vitro DNA replication and transcription of the Ad core. We have recently reported that TAF-I binds to major core protein VII and forms DNA-protein VII-TAF-I ternary complexes in vitro and in vivo. Further to understand the mechanism of remodeling of the Ad core, we characterized the interaction mode between the precursor of protein VII (pre-VII) and either DNA or TAF-I by means of biochemical and biophysicochemical methods. We found that a major binding region of pre-VII with both DNA and the acidic carboxyl-terminal region of TAF-I lies in the arginine-rich region of pre-VII. Both amino-terminal and carboxyl-terminal regions of pre-VII without the arginine-rich region directly bound to DNA and supported the DNA binding activity of the arginine-rich region. A TAF-I mutant protein lacking the acidic carboxyl-terminal region bound preferentially to the carboxyl-terminal region of pre-VII containing the arginine-rich region rather than the amino-terminal region of pre-VII. Thus, DNA interacted with the entire region of pre-VII, while TAF-I bound preferentially to the carboxyl-terminal region of pre-VII. This binding mode suggests the formation of the ternary complexes among DNA, protein VII, and TAF-I. On the basis of the binding modes in binary systems, we discussed the remodeling mechanism of the Ad core in early phases of infection.

Microcin B17 (MccB17) is a DNA gyrase poison; in previous work, this bacterial toxin was found to slowly and incompletely inhibit the reactions of supercoiling and relaxation of DNA by gyrase and to stabilize the cleavage complex, depending on the presence of ATP and the DNA topology. We now show that the action of MccB17 on the gyrase ATPase reaction and cleavage complex formation requires a linear DNA fragment of more than 150 base pairs. MccB17 is unable to stimulate the ATPase reaction by stabilizing the weak interactions between short linear DNA fragments (70 base pairs or less) and gyrase, in contrast with the quinolone ciprofloxacin. However, MccB17 can affect the ATP-dependent relaxation of DNA by gyrase lacking its DNA-wrapping or ATPase domains. From these findings, we propose a mode of action of MccB17 requiring a DNA molecule long enough to allow the transport of a segment through the DNA gate of the enzyme. Furthermore, we suggest that MccB17 may trap a transient intermediate state of the gyrase reaction present only during DNA strand passage and enzyme turnover. The proteolytic signature of MccB17 from trypsin treatment of the full enzyme requires DNA and ATP and shows a protection of the C-terminal 47-kDa domain of gyrase, indicating the involvement of this domain in the toxin mode of action and consistent with its proposed role in the mechanism of DNA strand passage. We suggest that the binding site of MccB17 is in the C-terminal domain of GyrB.

Human replication protein A (hRPA), a heterotrimeric single-stranded DNA (ssDNA) binding protein, is required for many cellular pathways including DNA damage repair, recombination, and replication as well as the ATR-mediated DNA damage response. While extensive effort has been devoted to understanding the structural relationships between RPA and ssDNA, information is currently limited to the RPA domains, the trimerization core, and a partial cocrystal structure. In this work, we employed a mass spectrometric protein footprinting method of single amino acid resolution to investigate the interactions of the entire heterotrimeric hRPA with ssDNA. In particular, we monitored surface accessibility of RPA lysines with NHS-biotin modification in the contexts of the free protein and the nucleoprotein complex. Our results not only indicated excellent agreement with the available crystal structure data for RPA70 DBD-AB-ssDNA complex but also revealed new protein contacts in the nucleoprotein complex. In addition to two residues, K263 and K343 of p70, previously identified by cocrystallography as direct DNA contacts, we observed protection of five additional lysines (K183, K259, K489, K577, and K588 of p70) upon ssDNA binding to RPA. Three residues, K489, K577, and K588, are located in ssDNA binding domain C and are likely to establish the direct contacts with cognate DNA. In contrast, no ssDNA-contacting lysines were identified in DBD-D. In addition, two lysines, K183 and K259, are positioned outside the putative ssDNA binding cleft. We propose that the protection of these lysines could result from the RPA interdomain structural reorganization induced by ssDNA binding.

Double-stranded DNA is a therapeutic target for a variety of anticancer and antimicrobial drugs. Noncovalent interactions of small molecules with DNA usually occur via intercalation of planar compounds between adjacent base pairs or minor-groove recognition by extended crescent-shaped ligands. However, the dynamic and flexibility of the DNA platform provide a variety of conformations that can be targeted by structurally diverse compounds. Here, we propose a novel DNA-binding template for construction of new therapeutic candidates. Four bisphenylcarbazole derivatives, derived from the combined molecular architectures of known antitumor bisphenylbenzimidazoles and anti-infectious dicationic carbazoles, have been designed, and their interaction with DNA has been studied by a combination of biochemical and biophysical methods. The substitutions of the bisphenylcarbazole core with two terminal dimethylaminoalkoxy side chains strongly promote the interaction with DNA, to prevent the heat denaturation of the double helix. The deletion or the replacement of the dimethylamino-terminal groups with hydroxyl groups strongly decreased DNA interaction, and the addition of a third cationic side chain on the carbazole nitrogen reinforced the affinity of the compound for DNA. Although the bi- and tridentate molecules both derive from well-characterized DNA minor-groove binders, the analysis of their binding mode by means of circular and linear dichroism methods suggests that these compounds form intercalation complexes with DNA. Negative-reduced dichroism signals were recorded in the presence of natural DNA and synthetic AT and GC polynucleotides. The intercalation hypothesis was validated by unwinding experiments using topoisomerase I. Prominent gel shifts were observed with the di- and trisubstituted bisphenylcarbazoles but not with the uncharged analogues. These observations, together with the documented stacking properties of such molecules (components for liquid crystals), prompted us to investigate their binding to the human telomeric DNA sequence by means of biosensor surface plasmon resonance. Under conditions favorable to G4 formation, the title compounds showed only a modest interaction with the telomeric quadruplex sequence, comparable to that measured with a double-stranded oligonucleotide. Their sequence preference was explored by DNase I footprinting experiments from which we identified a composite set of binding sequences comprising short AT stretches and a few other mixed AT/GC blocks with no special AT character. The variety of the binding sequences possibly reflects the coexistence of distinct positioning of the chromophore in the intercalation sites. The bisphenylcarbazole unit represents an original pharmacophore for DNA recognition. Its branched structure, with two or three arms suitable to introduce a structural diversity, provides an interesting scaffold to built molecules susceptible to discriminate between the different conformations of nucleic acids.

HMGB1 (high mobility group B1) is a conserved chromosomal protein composed of two similar DNA binding domains (HMG box A and box B) linked by a short basic stretch to an acidic C-terminal tail of 30 residues. The acidic tail modulates the DNA binding properties of HMGB1, and its length differentiates the various HMGB family members. We synthesized a peptide that corresponds to the acidic tail in HMGB1 (T-peptide) and studied its binding to the single boxes and to the fragment corresponding to tailless HMGB1 (designated as AB(bt) fragment). CD spectroscopy showed that T-peptide stabilizes significantly the AB(bt) fragment and that the complex has an identical thermal stability as full-length HMGB1. Calorimetric and NMR data showed that T-peptide binds with a dissociation constant of 9 microM to box A and much more weakly to box B. (1)H-(15)N HSQC spectra of full-length HMGB1 and of the AB(bt) fragment are very similar; the small chemical shift differences that exist correspond to those residues of the AB(bt) fragment that were affected by the addition of the T-peptide. We conclude that the T-peptide mimics closely the acidic tail and that the basic stretch and the acidic tail form an extended and flexible segment. The tail interacts with specific residues in the boxes and shields them from other interactions.

The testis-determining factor SRY contains an HMG box DNA-bending domain. Human and murine factors (hSRY and mSRY, respectively) exhibit marked sequence divergence and are reported to differ markedly in DNA bending properties. Surprisingly, the combined application of time-resolved fluorescence resonance energy transfer (tr-FRET) and permutation gel electrophoresis demonstrates that the hSRY-DNA complex is more sharply bent than the murine complex and not less bent as previously reported. tr-FRET-based analyses of the distribution of end-to-end distances in the bent DNA-protein complexes further suggest that a broader range of DNA bend angles is populated in the murine ensemble than in the human ensemble. The two domains and their respective DNA complexes nevertheless exhibit similar thermodynamic stabilities. (1)H NMR spectra indicate analogous intercalation of distinct "cantilever" side chains (isoleucine or methionine) with subtle differences in induced DNA structure. Interchange of cantilevers does not affect DNA bending. That transgenic expression of either human or murine Sry in XX mice can confer a male somatic phenotype suggests that SRY-directed transcriptional regulation is robust to enhanced DNA bending and to changes in the precision of DNA bending. We propose that male-specific gene regulation requires DNA bending above a critical threshold set by architectural requirements of enhanceosome assembly.

To better understand origin recognition and initiation of DNA replication, we have examined by NMR complexes formed between the origin-binding domain of SV40 T antigen (T-ag-obd), the initiator pro...

The cAMP receptor protein (CRP) of Escherichia coli is a transcription factor. The affinity of CRP for a specific DNA sequence is significantly enhanced as a consequence of the binding of the allosteric effector, cAMP. The hinge region, particularly residues 136 and 138, connecting the cAMP and DNA binding domains of CRP has been proposed to play essential roles in transmitting the allosteric signals. To probe the specific role of residue 138, eight D138 mutants and wild-type CRP were tested for their ability to bind the lac26 and gallac26 promoter sequences in this study. A correlation was established between DNA binding affinity and side chain solvation free energy, namely, an increasing specific DNA affinity with an increasing hydrophilicity of the side chain of residue 138. In addition, a linear correlation was found between DNA binding affinity and the energetics of subunit assembly. The ability of CRP to distinguish between cAMP and cGMP as an allosteric activator of DNA binding is weakened with higher energetics of subunit assembly. This correlation indicates that quaternary constraint leads to a constraint of the DNA binding domain. This observation is consistent with the concept that an optimum quaternary structural constraint is important in CRP exhibiting its allosteric properties. The stability of CRP was monitored by Trp fluorescence and circular dichroism in the presence of guanidine hydrochloride. These spectroscopic data revealed nonidentical denaturation profiles. Since the Trp residues are located exclusively in the beta-roll cyclic nucleotide binding domain, the denaturation profiles reveal the stability of the beta-roll structure. This study produces another linear correlation between DNA binding affinity in the presence of cAMP and cGMP and the stability of the beta-roll; namely, the stability of the beta-roll structure leads to a decrease in DNA binding affinity. All these correlations indicate the importance of structural stability and dynamics in the ability of CRP to manifest its intrinsic allosteric properties.