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Helicase enzymes translocate along an RNA or DNA template with a defined polarity to unwind, separate, or remodel duplex strands for a variety of genome maintenance processes. Helicase mutations are commonly associated with a variety of diseases including aging, cancer, and neurodegeneration. Biochemical characterization of these enzymes has provided a wealth of information on the kinetics of unwinding and substrate preferences, and several high-resolution structures of helicases alone and bound to oligonucleotides have been solved. Together, they provide mechanistic insights into the structural translocation and unwinding orientations of helicases. However, these insights rely on structural inferences derived from static snapshots. Instead, continued efforts should be made to combine structure and kinetics to better define active translocation orientations of helicases. This review explores many of the biochemical and biophysical methods utilized to map helicase binding orientation to DNA or RNA substrates and includes several time-dependent methods to unequivocally map the active translocation orientation of these enzymes to better define the active leading and trailing faces.

Several decades of research have identified Mcm10 hanging around the replisome making several critical contacts with a number of proteins but with no real disclosed function. Recently, the O'Donnell laboratory has been better able to map the interactions of Mcm10 with a larger Cdc45/GINS/MCM (CMG) unwinding complex placing it at the front of the replication fork. They have shown biochemically that Mcm10 has the impressive ability to strip off single-strand binding protein (RPA) and reanneal complementary DNA strands. This has major implications in controlling DNA unwinding speed as well as responding to various situations where fork reversal is needed. This work opens up a number of additional facets discussed here revolving around accessing the DNA junction for different molecular purposes within a crowded replisome. Abbreviations: alt-NHEJ: Alternative Nonhomologous End-Joining; CC: Coli-Coil motif; CMG: Cdc45/GINS/MCM2-7; CMGM: Cdc45/GINS/Mcm2-7/Mcm10; CPT: Camptothecin; CSB: Cockayne Syndrome Group B protein; CTD: C-Terminal Domain; DSB: Double-Strand Break; DSBR: Double-Strand Break Repair; dsDNA: Double-Stranded DNA; GINS: go-ichi-ni-san, Sld5-Psf1-Psf2-Psf3; HJ Dis: Holliday Junction dissolution; HJ Res: Holliday Junction resolution; HR: Homologous Recombination; ICL: Interstrand Cross-Link; ID: Internal Domain; MCM: Minichromosomal Maintenance; ND: Not Determined; NTD: N-Terminal Domain; PCNA: Proliferating Cell Nuclear Antigen; RPA: Replication Protein A; SA: Strand Annealing; SE: Strand Exchange; SEW: Steric Exclusion and Wrapping; ssDNA: Single-Stranded DNA; TCR: Transcription-Coupled Repair; TOP1: Topoisomerase.

Helicase regulation is vital for replisome progression, where the helicase enzyme functions to unwind duplex DNA and aids in the coordination of replication fork activities. Currently, mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro. To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified SEW-deficient mutants of the bacterial replicative helicase DnaB. We discovered that these four SEW mutations stabilize constricted states, with more fully constricted mutants having a generally greater impact on genomic stress, suggesting a dynamic model for helicase regulation that involves both excluded strand interactions and conformational states. These dnaB mutations result in increased DNA damage and chromosome complexity, less stable genomes, and ultimately less viable and fit strains. Notably, while two mutations stabilized fully constricted states, they have distinct effects on genomic stability, suggesting a complex relationship between helicase regulation mechanisms and faithful, efficient DNA replication. This work explores the genomic impacts of helicase dysregulation in vivo, supporting a combined dynamic regulatory mechanism involving SEW and conformational changes and relates current mechanistic understanding to functional helicase behavior.

During DNA replication, the presence of 8-oxoguanine (8-oxoG) lesions in the template strand cause the high-fidelity (HiFi) DNA polymerase (Pol) to stall. An early response to 8-oxoG lesions involves ‘on-the-fly’ translesion synthesis (TLS), in which a specialized TLS Pol is recruited and replaces the stalled HiFi Pol for bypass of the lesion. The length of TLS must be long enough for effective bypass, but it must also be regulated to minimize replication errors by the TLS Pol. The exact position where the TLS Pol ends and the HiFi Pol resumes (i.e. the length of the TLS patch) has not been described. We use steady-state and pre-steady-state kinetic assays to characterize lesion bypass intermediates formed by different archaeal polymerase holoenzyme complexes that include PCNA123 and RFC. After bypass of 8-oxoG by TLS PolY, products accumulate at the template position three base pairs beyond the lesion. PolY is catalytically poor for subsequent extension from this +3 position beyond 8-oxoG, but this inefficiency is overcome by rapid extension of HiFi PolB1. The reciprocation of Pol activities at this intermediate indicates a defined position where TLS Pol extension is limited and where the DNA substrate is handed back to the HiFi Pol after bypass of 8-oxoG.

Helicase regulation involves modulation of unwinding speed to maintain coordination of DNA replication fork activities and is vital for replisome progression. Currently, mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro. To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified SEW-deficient mutants of the bacterial replicative helicase DnaB. We discovered that these four SEW mutations stabilize constricted states, with more fully constricted mutants having a generally greater impact on genomic stress, suggesting a dynamic model for helicase regulation that involves both excluded strand interactions and conformational states. These dnaB mutations result in increased chromosome complexities, less stable genomes, and ultimately less viable and fit strains. Specifically, dnaB:mut strains present with increased mutational frequencies without significantly inducing SOS, consistent with leaving single-strand gaps in the genome during replication that are subsequently filled with lower fidelity. This work explores the genomic impacts of helicase dysregulation in vivo, supporting a combined dynamic regulatory mechanism involving a spectrum of DnaB conformational changes and relates current mechanistic understanding to functional helicase behavior at the replication fork., Author summary DNA replication is a vital biological process, and the proteins involved are structurally and functionally conserved across all domains of life. As our fundamental knowledge of genes and genetics grows, so does our awareness of links between acquired genetic mutations and disease. Understanding how genetic material is replicated accurately and efficiently and with high fidelity is the foundation to identifying and solving genome-based diseases. E. coli are model organisms, containing core replisome proteins, but lack the complexity of the human replication system, making them ideal for investigating conserved replisome behaviors. The helicase enzyme acts at the forefront of the replication fork to unwind the DNA helix and has also been shown to help coordinate other replisome functions. In this study, we examined specific mutations in the helicase that have been shown to regulate its conformation and speed of unwinding. We investigate how these mutations impact the growth, fitness, and cellular morphology of bacteria with the goal of understanding how helicase regulation mechanisms affect an organism’s ability to survive and maintain a stable genome.

Replicative hexameric helicases are thought to unwind duplex DNA by steric exclusion (SE) where one DNA strand is encircled by the hexamer and the other is excluded from the central channel. However, interactions with the excluded strand on the exterior surface of hexameric helicases have also been shown to be important for DNA unwinding, giving rise to the steric exclusion and wrapping (SEW) model. For example, the archaeal Sulfolobus solfataricus minichromosome maintenance (SsoMCM) helicase has been shown to unwind DNA via a SEW mode to enhance unwinding efficiency. Using single-molecule FRET, we now show that the analogous Escherichia coli (Ec) DnaB helicase also interacts specifically with the excluded DNA strand during unwinding. Mutation of several conserved and positively charged residues on the exterior surface of EcDnaB resulted in increased interaction dynamics and states compared with wild type. Surprisingly, these mutations also increased the DNA unwinding rate, suggesting that electrostatic contacts with the excluded strand act as a regulator for unwinding activity. In support of this, experiments neutralizing the charge of the excluded strand with a morpholino substrate instead of DNA also dramatically increased the unwinding rate. Of note, although the stability of the excluded strand was nearly identical for EcDnaB and SsoMCM, these enzymes are from different superfamilies and unwind DNA with opposite polarities. These results support the SEW model of unwinding for EcDnaB that expands on the existing SE model of hexameric helicase unwinding to include contributions from the excluded strand to regulate the DNA unwinding rate.

Before leaving the house, it is a good idea to check for road closures that may affect the morning commute. Otherwise, one may encounter significant delays arriving at the destination. While this is commonly true, motorists may be able to consult a live interactive traffic map and pick an alternate route or detour to avoid being late. However, this is not the case if one needs to catch the train which follows a single track to the terminus; if something blocks the track, there is a delay. Such is the case for the DNA replisome responsible for copying the genetic information that provides the recipe of life. When the replication machinery encounters a DNA roadblock, the outcome can be devastating if the obstacle is not overcome in an efficient manner. Fortunately, the cell's DNA synthesis apparatus can bypass certain DNA obstructions, but the mechanism(s) are still poorly understood. Very recently, two papers from the O'Donnell lab, one structural (Georgescu et al., 2017 [1]) and the other biochemical (Langston and O'Donnell, 2017 [2]), have challenged the conventional thinking of how the replicative CMG helicase is arranged on DNA, unwinds double-stranded DNA, and handles barricades in its path. These new findings raise important questions in the search for mechanistic insights into how DNA is copied, particularly when the replication machinery encounters a roadblock.

The ability of the replisome to seamlessly coordinate both high fidelity and translesion DNA synthesis requires a means to regulate recruitment and binding of enzymes from solution. Co-occupancy of multiple DNA polymerases within the replisome has been observed primarily in bacteria and is regulated by posttranslational modifications in eukaryotes, and both cases are coordinated by the processivity clamp. Because of the heterotrimeric nature of the PCNA clamp in some archaea, there is potential to occupy and regulate specific polymerases at defined subunits. In addition to specific PCNA and polymerase interactions (PIP site), we have now identified and characterized a novel protein contact between the Y-family DNA polymerase and the B-family replication polymerase (YB site) bound to PCNA and DNA from Sulfolobus solfataricus. These YB contacts are essential in forming and stabilizing a supraholoenzyme (SHE) complex on DNA, effectively increasing processivity of DNA synthesis. The SHE complex can not only coordinate polymerase exchange within the complex but also provides a mechanism for recruitment of polymerases from solution based on multiequilibrium processes. Our results provide evidence for an archaeal PCNA ‘tool-belt’ recruitment model of multienzyme function that can facilitate both high fidelity and translesion synthesis within the replisome during DNA replication.

DNA replication requires that the duplex genomic DNA strands be separated; a function that is implemented by ring-shaped hexameric helicases in all Domains. Helicases are composed of two domains, an N- terminal DNA binding domain (NTD) and a C- terminal motor domain (CTD). Replication is controlled by loading of helicases at origins of replication, activation to preferentially encircle one strand, and then translocation to begin separation of the two strands. Using a combination of site-specific DNA footprinting, single-turnover unwinding assays, and unique fluorescence translocation monitoring, we have been able to quantify the binding distribution and the translocation orientation of Saccharolobus (formally Sulfolobus) solfataricus MCM on DNA. Our results show that both the DNA substrate and the C-terminal winged-helix (WH) domain influence the orientation but that translocation on DNA proceeds N-first.

There are several DNA helicases involved in seemingly overlapping aspects of homologous and homoeologous recombination. Mutations of many of these helicases are directly implicated in genetic diseases including cancer, rapid aging, and infertility. MCM8/9 are recent additions to the catalog of helicases involved in recombination, and so far, the evidence is sparse, making assignment of function difficult. Mutations in MCM8/9 correlate principally with primary ovarian failure/insufficiency (POF/POI) and infertility indicating a meiotic defect. However, they also act when replication forks collapse/break shuttling products into mitotic recombination and several mutations are found in various somatic cancers. This review puts MCM8/9 in context with other replication and recombination helicases to narrow down its genomic maintenance role. We discuss the known structure/function relationship, the mutational spectrum, and dissect the available cellular and organismal data to better define its role in recombination.

Hexameric DNA helicases involved in the separation of duplex DNA at the replication fork have a universal architecture but have evolved from two separate protein families. The consequences are that the regulation, translocation polarity, strand specificity, and architectural orientation varies between phage/bacteria to that of archaea/eukaryotes. Once assembled and activated for single strand DNA translocation and unwinding, the DNA polymerase couples tightly to the helicase forming a robust replisome complex. However, this helicase-polymerase interaction can be challenged by various forms of endogenous or exogenous agents that can stall the entire replisome or decouple DNA unwinding from synthesis. The consequences of decoupling can be severe, leading to a build-up of ssDNA requiring various pathways for replication fork restart. All told, the hexameric helicase sits prominently at the front of the replisome constantly responding to a variety of obstacles that require transient unwinding/reannealing, traversal of more stable blocks, and alternations in DNA unwinding speed that regulate replisome progression.

The MCM8/9 complex is implicated in aiding fork progression and facilitating homologous recombination (HR) in response to several DNA damage agents. MCM9 itself is an outlier within the MCM family containing a long C-terminal extension (CTE) comprising 42% of the total length, but with no known functional components and high predicted disorder. In this report, we identify and characterize two unique motifs within the primarily unstructured CTE that are required for localization of MCM8/9 to sites of mitomycin C (MMC) induced DNA damage. First, an unconventional ‘bipartite-like’ nuclear localization (NLS) motif consisting of two positively charged amino acid stretches separated by a long intervening sequence is required for the nuclear import of both MCM8 and MCM9. Second, a variant of the BRC motif (BRCv), similar to that found in other HR helicases, is necessary for localization to sites of MMC damage. The MCM9-BRCv directly interacts with and recruits RAD51 downstream to MMC-induced damage to aid in DNA repair. Patient lymphocytes devoid of functional MCM9 and discrete MCM9 knockout cells have a significantly impaired ability to form RAD51 foci after MMC treatment. Therefore, the disordered CTE in MCM9 is functionally important in promoting MCM8/9 activity and in recruiting downstream interactors; thus, requiring full length MCM9 for proper DNA repair.

Helicases are proposed to unwind dsDNA primarily by translocating on one strand to sterically exclude and separate the two strands. Hexameric helicases in particular have been shown to encircle one strand while physically excluding the other strand. In this article, we will detail experimental methods used to validate specific interactions with the excluded strand on the exterior surface of hexameric helicases. Both qualitative and quantitative methods are described to identify an excluded strand interaction, determine the exterior interacting residues, and measure the dynamics of binding. The implications of exterior interactions with the nontranslocating strand are discussed and include forward unwinding stabilization, regulation of the unwinding rate, and DNA damage sensing.

Mutations in the c10orf2 gene encoding the human mitochondrial DNA replicative helicase Twinkle are linked to several rare genetic diseases characterized by mitochondrial defects. In this study, we have examined the catalytic activity of Twinkle helicase on model replication fork and DNA repair structures. Although Twinkle behaves as a traditional 5′ to 3′ helicase on conventional forked duplex substrates, the enzyme efficiently dissociates D-loop DNA substrates irrespective of whether it possesses a 5′ or 3′ single-stranded tailed invading strand. In contrast, we report for the first time that Twinkle branch-migrates an open-ended mobile three-stranded DNA structure with a strong 5′ to 3′ directionality preference. To determine how well Twinkle handles potential roadblocks to mtDNA replication, we tested the ability of the helicase to unwind substrates with site-specific oxidative DNA lesions or bound by the mitochondrial transcription factor A. Twinkle helicase is inhibited by DNA damage in a unique manner that is dependent on the type of oxidative lesion and the strand in which it resides. Novel single molecule FRET binding and unwinding assays show an interaction of the excluded strand with Twinkle as well as events corresponding to stepwise unwinding and annealing. TFAM inhibits Twinkle unwinding, suggesting other replisome proteins may be required for efficient removal. These studies shed new insight on the catalytic functions of Twinkle on the key DNA structures it would encounter during replication or possibly repair of the mitochondrial genome and how well it tolerates potential roadblocks to DNA unwinding.

The archaeal minichromosomal maintenance (MCM) helicase from Sulfolobus solfataricus (SsoMCM) is a model for understanding structural and mechanistic aspects of DNA unwinding. Although interactions of the encircled DNA strand within the central channel provide an accepted mode for translocation, interactions with the excluded strand on the exterior surface have mostly been ignored with regard to DNA unwinding. We have previously proposed an extension of the traditional steric exclusion model of unwinding to also include significant contributions with the excluded strand during unwinding, termed steric exclusion and wrapping (SEW). The SEW model hypothesizes that the displaced single strand tracks along paths on the exterior surface of hexameric helicases to protect single-stranded DNA (ssDNA) and stabilize the complex in a forward unwinding mode. Using hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance MS, we have probed the binding sites for ssDNA, using multiple substrates targeting both the encircled and excluded strand interactions. In each experiment, we have obtained >98.7% sequence coverage of SsoMCM from >650 peptides (5–30 residues in length) and are able to identify interacting residues on both the interior and exterior of SsoMCM. Based on identified contacts, positively charged residues within the external waist region were mutated and shown to generally lower DNA unwinding without negatively affecting the ATP hydrolysis. The combined data globally identify binding sites for ssDNA during SsoMCM unwinding as well as validating the importance of the SEW model for hexameric helicase unwinding.

Contains fulltext : 193270.pdf (Publisher’s version ) (Open Access) The clamp protein (gp45) of the DNA polymerase III of the bacteriophage T4 is known to bind to DNA and stay attached to it in order to facilitate the process of DNA copying by the polymerase. As part of a project aimed at developing new biomimetic data-encoding systems we have investigated the binding of gp45 to synthetic polymers, that is, rigid, helical polyisocyanopeptides. Molecular modelling studies suggest that the clamp protein may interact with the latter polymers. Experiments aimed at verifying these interactions are presented and discussed.

Notwithstanding numerous published structures of RNA Polymerase II (Pol II), structural details of Pol II engaging a complete nucleic acid scaffold have been lacking. Here, we report the structures of TFIIF stabilized transcribing Pol II complexes, revealing the upstream duplex and full transcription bubble. The upstream duplex lies over a wedge-shaped loop from Rpb2 that engages its minor groove, providing part of the structural framework for DNA tracking during elongation. At the upstream transcription bubble fork, rudder and fork loop-1 residues spatially coordinate strand annealing and the nascent RNA transcript. At the downstream fork, a network of Pol II interactions with the non-template strand forms a rigid domain with the Trigger Loop (TL), allowing visualization of its open state. Overall, our observations suggest that “open/closed” conformational transitions of the TL may be linked to interactions with the non-template strand, possibly in a synchronized ratcheting manner conducive to polymerase translocation.

The ability for DNA polymerases (Pols) to overcome a variety of obstacles in its path to maintain genomic stability during replication is a complex endeavor. It requires the coordination of multiple Pols with differing specificities through molecular control and access to the replisome. Although a number of contacts directly between Pols and accessory proteins have been identified, forming the basis of a variety of holoenzyme complexes, the dynamics of Pol active site substitutions remain uncharacterized. Substitutions can occur externally by recruiting new Pols to replisome complexes through an "exchange" of enzyme binding or internally through a "switch" in the engagement of DNA from preformed associated enzymes contained within supraholoenzyme complexes. Models for how high fidelity (HiFi) replication Pols can be substituted by translesion synthesis (TLS) Pols at sites of damage during active replication will be discussed. These substitution mechanisms may be as diverse as the number of Pol families and types of damage; however, common themes can be recognized across species. Overall, Pol substitutions will be controlled by explicit protein contacts, complex multiequilibrium processes, and specific kinetic activities. Insight into how these dynamic processes take place and are regulated will be of utmost importance for our greater understanding of the specifics of TLS as well as providing for future novel chemotherapeutic and antimicrobial strategies.

In processive catalysis, a catalyst binds to a substrate and remains bound as it performs several consecutive reactions, as exemplified by DNA polymerases. Processivity is essential in nature and is often mediated by a clamp-like structure that physically tethers the catalyst to its (polymeric) template. In the case of the bacteriophage T4 replisome, a dedicated clamp protein acts as a processivity mediator by encircling DNA and subsequently recruiting its polymerase. Here we use this DNA-binding protein to construct a biohybrid catalyst. Conjugation of the clamp protein to a chemical catalyst with sequence-specific oxidation behaviour formed a catalytic clamp that can be loaded onto a DNA plasmid. The catalytic activity of the biohybrid catalyst was visualized using a procedure based on an atomic force microscopy method that detects and spatially locates oxidized sites in DNA. Varying the experimental conditions enabled switching between processive and distributive catalysis and influencing the sliding direction of this rotaxane-like catalyst.

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.

DNA replication polymerases have the inherent ability to faithfully and rapidly copy a DNA template according to precise Watson-Crick base pairing. The primary B-family DNA replication polymerase (Dpo1) in the hyperthermophilic archaeon, Sulfolobus solfataricus, is shown here to possess a remarkable DNA stabilizing ability for maintaining weak base pairing interactions to facilitate primer extension. This thermal stabilization by Dpo1 allowed for template-directed synthesis at temperatures more than 30 °C above the melting temperature of naked DNA. Surprisingly, Dpo1 also displays a competing terminal deoxynucleotide transferase (TdT) activity unlike any other B-family DNA polymerase. Dpo1 is shown to elongate single-stranded DNA in template-dependent and template-independent manners. Experiments with different homopolymeric templates indicate that initial deoxyribonucleotide incorporation is complementary to the template. Rate-limiting steps that include looping back and annealing to the template allow for a unique template-dependent terminal transferase activity. The multiple activities of this unique B-family DNA polymerase make this enzyme an essential component for DNA replication and DNA repair for the maintenance of the archaeal genome at high temperatures.

DNA polymerases are essential enzymes in all domains of life for both DNA replication and repair. The primary DNA replication polymerase from Sulfolobus solfataricus (SsoDpo1) has been shown previously to provide the necessary polymerization speed and exonuclease activity to replicate the genome accurately. We find that this polymerase is able to physically associate with itself to form a trimer and that this complex is stabilized in the presence of DNA. Analytical gel filtration and electrophoretic mobility shift assays establish that initially a single DNA polymerase binds to DNA followed by the cooperative binding of two additional molecules of the polymerase at higher concentrations of the enzyme. Protein chemical crosslinking experiments show that these are specific polymerase-polymerase interactions and not just separate binding events along DNA. Isothermal titration calorimetry and fluorescence anisotropy experiments corroborate these findings and show a stoichiometry where three polymerases are bound to a single DNA substrate. The trimeric polymerase complex significantly increases both the DNA synthesis rate and the processivity of SsoDpo1. Taken together, these results suggest the presence of a trimeric DNA polymerase complex that is able to synthesize long DNA strands more efficiently than the monomeric form.

The archaeal minichromosome maintenance protein MCM forms a homohexameric complex that functions as the DNA replicative helicase and serves as a model system for its eukaryotic counterpart. Here, we applied single molecule fluorescence resonance energy transfer methods to probe the substrate specificity and binding mechanism of MCM from the hyperthermophilic Archaea Sulfolobus solfataricus on various DNA substrates. S. solfataricus MCM displays a binding preference for forked substrates relative to partial or full duplex substrates. Moreover, the nature of MCM binding to Y-shaped substrates is distinct in that MCM loads on the 3'-tail while interacting with the 5'-tail likely via the MCM surface. These results provide the first elucidation of a dynamic nature of interaction between a ring-shaped helicase interacting with an opposing single-stranded DNA tail. This interaction contributes to substrate selectivity and increases the stability of the forked DNA-MCM complex, with possible implications for the MCM unwinding mechanism.

The polymerase (gp43) processivity during T4 replisome mediated DNA replication has been investigated. The size of the Okazaki fragments remains constant over a wide range of polymerase concentrations. A dissociation rate constant of ≈0.0013 sec -1 was measured for the polymerases from both strands, consistent with highly processive replication on both the leading and lagging strands. This processive replication, however, can be disrupted by a catalytically inactive mutant D408N gp43 that retains normal affinity for DNA and the clamp. The inhibition kinetics fit well to an active exchange model in which the mutant polymerase (the polymerase trap) displaces the replicating polymerase. This kinetic model was further strengthened by the observation that the sizes of both the Okazaki fragments and the extension products on a primed M13mp18 template were reduced in the presence of the mutant polymerase. The effects of the trap polymerase therefore suggest a dynamic processivity of the polymerase during replication, namely, a solution/replisome polymerase exchange takes place without affecting continued DNA synthesis. This process mimics the polymerase switching recently suggested during the translesion DNA synthesis, implies the multiple functions of the clamp in replication, and may play a potential role in overcoming the replication barriers by the T4 replisome.

Transient kinetic analyses further support the role of the clamp-loader in bacteriophage T4 as a catalyst which loads the clamp onto DNA through the sequential hydrolysis of two molecules of ATP before and after addition of DNA. Additional rapid-quench and pulse-chase experiments have documented this stoichiometry. The events of ATP hydrolysis have been related to the opening/closing of the clamp protein through fluorescence resonance energy transfer (FRET). In the absence of a hydrolysable form of ATP, the distance across the subunit interface of the clamp does not increase as measured by intramolecular FRET, suggesting gp45 cannot be loaded onto DNA. Therefore, ATP hydrolysis by the clamp-loader appears to open the clamp wide enough to encircle DNA easily. Two additional molecules of ATP then are hydrolyzed to close the clamp onto DNA. The presence of an intermolecular FRET signal indicated that the dissociation of the clamp-loader from this complex occurred after guiding the polymerase onto the correct face of the clamp bound to DNA. The final holoenzyme complex consists of the clamp, DNA, and the polymerase. Although this sequential assembly mechanism can be generally applied to most other replication systems studied to date, the specifics of ATP utilization seem to vary across replication systems.

The bacteriophage T4 replication complex is composed of eight proteins that function together to replicate DNA. This replisome can be broken down into four basic units: a primosome composed of gp41, gp61, and gp59; a leading strand holoenzyme composed of gp43, gp44/62, and gp45; a lagging strand holoenzyme; and a single strand binding protein polymer. These units interact further to form the complete replisome. The leading and lagging strand polymerases are physically linked in the presence of DNA or an active replisome. The region of interaction was mapped to an extension of the finger domain, such that Cys-507 of one subunit is in close proximity to Cys-507 of a second subunit. The leading strand polymerase and the primosome also associate, such that gp59 mediates the contact between the two complexes. Binding of gp43 to the primosome complex causes displacement of gp32 from the gp59.gp61.gp41 primosome complex. The resultant species is a complex of proteins that may allow coordinated leading and lagging strand synthesis, helicase DNA unwinding activity, and polymerase nucleotide incorporation.

Single-molecule studies reveal that a ring-like enzyme that encircles and 'slides' along one strand of duplex DNA, separating it from the other strand, overcomes molecular barriers in its path by transiently opening its ring. See Article p.205 The replicative helicase of simian virus 40 (SV40), the large T antigen (T-Ag), was thought to form a double hexamer that passed duplex DNA through the centre, forcing the unwound single-stranded DNA out the sides. Johannes Walter and colleagues now show that this model is incorrect, as the functional form is a single hexamer. Surprisingly, proteins bound to DNA do not form a barrier to movement of T-Ag, which is able to crack open the hexamer to bypass such protein roadblocks.

Most organisms encode for multiple DNA polymerases with similar substrate affinities, but vastly different fidelities. Proper genomic maintenance by the high fidelity (PolB1) and lesion bypass polymerases (PolY) from Sulfolobus solfataricus involves a complex solution equilibria of protein complexes and specific recognition of appropriate DNA substrates. Using isothermal titration calorimetry (ITC) and temperature dependent electrophoretic mobility shift assays (tEMSAs), we have found differences in oligomeric assemblies of Dpo1 and Dpo4 on DNA that include unusually strong temperature dependence changes in heat capacity (ΔCp), which switch from positive to negative values as temperature increases over a 60°C range. The thermodynamic data suggests that binding of PolB1 to DNA is favored over PolY by changes in solution multiequilibria (monomer-oligomer) with temperature that influence ΔCp values. We have also followed polymerase exchange events between PolB1 and PolY and themselves and with the sliding clamp, PCNA, during active DNA synthesis using ensemble kinetic and fluorescence resonance energy transfer (FRET) assays. Surprisingly, the assembled PolB1 holoenzyme complex synthesizes DNA distributively and with low processivity, unlike most other well-characterized DNA polymerase holoenzyme complexes. Exchange between polymerases is temperature and concentration dependent process that is orchestrated by several contacts with PCNA. Simultaneous binding of PolB1 and PolY1 to PCNA allows for dynamic exchange of polymerase active sites during replication and lesion bypass synthesis. Taken together, our results distinguish between specific thermodynamic parameters including temperature dependent coupled equilibria and structural complementary; kinetic processes; and protein contacts that direct binding for uninterrupted but dynamic DNA replication and repair processes at high temperatures.

Assembly of DNA replication systems requires the coordinated actions of many proteins. The multiprotein complexes formed as intermediates on the pathway to the final DNA polymerase holoenzyme have been shown to have distinct structures relative to the ground-state structures of the individual proteins. By using a variety of solution-phase techniques, we have elucidated additional information about the solution structure of the bacteriophage T4 holoenzyme. Photocross-linking and mass spectrometry were used to demonstrate interactions between I107C of the sliding clamp and the DNA polymerase. Fluorescence resonance energy transfer, analytical ultracentrifugation, and isothermal titration calorimetry measurements were used to demonstrate that the C terminus of the DNA polymerase can interact at two distinct locations on the sliding clamp. Both of these binding modes may be used during holoenzyme assembly, but only one of these binding modes is found in the final holoenzyme. Present and previous solution interaction data were used to build a model of the holoenzyme that is consistent with these data.

The bacteriophage T4 DNA replisome is a complex dynamic system employing a variety of proteins to orchestrate the synthesis of DNA on both the leading and lagging strands. Assembly of the protein complexes responsible for DNA synthesis and priming requires the coordination of transient biomolecular interactions. This interplay of proteins has been dissected through the use of small molecules including fluorescent probes and crosslinkers, enabling the development of a complex dynamic structural and kinetic model for DNA polymerase holoenzyme assembly and primosome formation.

The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 binds to this complex to generate the holoenzyme in which gp45 acts to topologically link gp43 to DNA, effectively increasing the processivity of DNA replication. Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly. By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring at one of the three subunit interfaces. On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP. Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation. The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.

Rho-associated coiled coil containing protein kinase (Rho-kinase or Rock) is a well-defined determinant of actin organization and dynamics in most animal cells characterized to date. One of the primary effectors of Rock is non-muscle myosin II. Activation of Rock results in increased contractility of myosin II and subsequent changes in actin architecture and cell morphology. The regulation of Rock is thought to occur via autoinhibition of the kinase domain via intramolecular interactions between the N-terminus and the C-terminus of the kinase. This autoinhibited state can be relieved via proteolytic cleavage, binding of lipids to a Pleckstrin Homology domain near the C-terminus, or binding of GTP-bound RhoA to the central coiled-coil region of Rock. Recent work has identified the Shroom family of proteins as an additional regulator of Rock either at the level of cellular distribution or catalytic activity or both. The Shroom-Rock complex is conserved in most animals and is essential for the formation of the neural tube, eye, and gut in vertebrates. To address the mechanism by which Shroom and Rock interact, we have solved the structure of the coiled-coil region of Rock that binds to Shroom proteins. Consistent with other observations, the Shroom binding domain is a parallel coiled-coil dimer. Using biochemical approaches, we have identified a large patch of residues that contribute to Shrm binding. Their orientation suggests that there may be two independent Shrm binding sites on opposing faces of the coiled-coil region of Rock. Finally, we show that the binding surface is essential for Rock colocalization with Shroom and for Shroom-mediated changes in cell morphology.

Template-dependent and template-independent nucleotidyl transfer reactions are fundamentally important in the maintenance of the genome as well as for gene expression in all organisms and viruses. These reactions are conserved and involve the condensation of an incoming nucleotide triphosphate at the 3′ hydroxyl of the growing oligonucleotide chain with concomitant release of pyrophosphate. DNA polymerase I (Pol I) isolated from E. coli extracts was initially characterized in in vitro reactions well over 50 years ago by the seminal work of Arthur Kornberg’s laboratory (Kornberg 1957; Lehman et al. 1958; Bessman et al. 1958). Inspired by this work, the discovery of a DNA-dependent RNA polymerase quickly followed in 1960 from a variety of researchers including Samuel Weiss (Weiss and Gladstone 1959), Jerald Hurwitz (Hurwitz et al. 1960), Audrey Stevens (Stevens 1960), and James Bonner (Huang et al. 1960). These early enzymatic characterizations of DNA-dependent deoxyribonucleotides and ribonucleotide incorporations gave credibility both to Watson and Crick’s DNA double helix model (Watson and Crick 1953) and the transcription operon model proposed by Francois Jacob and Jacques Monod (Jacob and Monod 1961).

The assembly and enzymatic ability of the replication DNA polymerase holoenzyme from Sulfolobus solfataricus (Sso) was investigated using presteady-state fluorescence resonance energy transfer assays coupled with functional and structural studies. Kinetic experiments reveal that ATP binding to replication factor C (RFC) is sufficient for loading the heterotrimeric PCNA123 [proliferating cell nuclear antigen (PCNA)] clamp onto DNA that includes a rate-limiting conformational rearrangement of the complex. ATP hydrolysis is required for favorable recruitment and interactions with the replication polymerase (PolB1) that most likely include clamp closing and RFC dissociation. Surprisingly, the assembled holoenzyme complex synthesizes DNA distributively and with low processivity, unlike most other well-characterized DNA polymerase holoenzyme complexes. We show that PolB1 repeatedly disengages from the DNA template, leaving PCNA123 behind. Interactions with a newly identified C-terminal PCNA-interacting peptide (PIP) motif on PolB1 specifically with PCNA2 are required for holoenzyme formation and continuous re-recruitment during synthesis. The extended tail-like structure of the C-terminal PIP motif in PolB1 is revealed alone and when bound to DNA using small-angle X-ray scattering allowing us to develop a model for the holoenzyme complex. This is the first detailed kinetic description of clamp loading and holoenzyme assembly in crenarchaea and has revealed a novel mode for dynamic processivity that occurs by a polymerase exchange mechanism. This work has important implications for processive DNA replication synthesis and also suggests a potential mechanism for polymerase switching to bypass lesions.

Hexameric helicases control both the initiation and the elongation phase of DNA replication. The toroidal structure of these enzymes provides an inherent challenge in the opening and loading onto DNA at origins, as well as the conformational changes required to exclude one strand from the central channel and activate DNA unwinding. Recently, high-resolution structures have not only revealed the architecture of various hexameric helicases but also detailed the interactions of DNA within the central channel, as well as conformational changes that occur during loading. This structural information coupled with advanced biochemical reconstitutions and biophysical methods have transformed our understanding of the dynamics of both the helicase structure and the DNA interactions required for efficient unwinding at the replisome.

The Shrm SD2 region contains a core that adopts a novel three-segmented dimer required for Rock binding. Conserved interfaces critical for Rock binding, ppMLC levels, and the formation of contractile cytoskeletal networks are identified. The complex is likely tetrameric, which suggests that conformational changes within SD2 are likely upon Rock binding., Shroom (Shrm) proteins are essential regulators of cell shape and tissue morpho­logy during animal development that function by interacting directly with the coiled-coil region of Rho kinase (Rock). The Shrm–Rock interaction is sufficient to direct Rock subcellular localization and the subsequent assembly of contractile actomyosin networks in defined subcellular locales. However, it is unclear how the Shrm–Rock interaction is regulated at the molecular level. To begin investigating this issue, we present the structure of Shrm domain 2 (SD2), which mediates the interaction with Rock and is required for Shrm function. SD2 is a unique three-segmented dimer with internal symmetry, and we identify conserved residues on the surface and within the dimerization interface that are required for the Rock–Shrm interaction and Shrm activity in vivo. We further show that these residues are critical in both vertebrate and invertebrate Shroom proteins, indicating that the Shrm–Rock signaling module has been functionally and molecularly conserved. The structure and biochemical analysis of Shrm SD2 indicate that it is distinct from other Rock activators such as RhoA and establishes a new paradigm for the Rock-mediated assembly of contractile actomyosin networks.

We have biochemically and kinetically characterized the polymerase and exonuclease activities of the third B-family polymerase (Dpo3) from the hyperthermophilic Crenarchaeon, Sulfolobus solfataricus (Sso). We have established through mutagenesis that despite incomplete sequence conservation, the polymerase and exonuclease active sites are functionally conserved in Dpo3. Using pre-steady-state kinetics, we can measure the fidelity of nucleotide incorporation by Dpo3 from the polymerase active site alone to be 10(3)-10(4) at 37 °C. The functional exonuclease proofreading active site will increase fidelity by at least 10(2), making Dpo3 comparable to other DNA polymerases in this family. Additionally, Dpo3's exonuclease activity is modulated by temperature, where a loss of promiscuous degradation activity can be attributed to a reorganization of the exonuclease domain when it is bound to primer-template DNA at high temperatures. Unexpectedly, the DNA binding affinity is weak compared with those of other DNA polymerases of this family. A comparison of the fidelity, polymerization kinetics, and associated functional exonuclease domain with those previously reported for other Sso polymerases (Dpo1 and Dpo4) illustrates that Dpo3 is a potential player in the proper maintenance of the archaeal genome.

The minichromosome maintenance (MCM) helicase complex is essential for the initiation and elongation of DNA replication in both the eukaryotic and archaeal domains. The archaeal homohexameric MCM helicase from Sulfolobus solfataricus serves as a model for understanding mechanisms of DNA unwinding. In this report, the displaced 5′-tail is shown to provide stability to the MCM complex on DNA and contribute to unwinding. Mutations in a positively charged patch on the exterior surface of the MCM hexamer destabilize this interaction, alter the path of the displaced 5′-tail DNA and reduce unwinding. DNA footprinting and single-molecule fluorescence experiments support a previously unrecognized wrapping of the 5′-tail. This mode of hexameric helicase DNA unwinding is termed the steric exclusion and wrapping (SEW) model, where the 3′-tail is encircled by the helicase while the displaced 5′-tail wraps around defined paths on the exterior of the helicase. The novel wrapping mechanism stabilizes the MCM complex in a positive unwinding mode, protects the displaced single-stranded DNA tail and prevents reannealing.

DNA replication is an essential process across all domains of life. Replicative helicases play an integral part in this process by unwinding the duplex DNA to make single-strand template strands available for duplication. While the steric exclusion model of unwinding, where one strand is encircled by the hexameric helicase and the other excluded from the central channel, is widely accepted, the complexities of this process remain unclear. Details of the helicases’ loading and unwinding mechanism(s) are continually being revealed. One such detail is the interaction and role played by the excluded strand.Our group has recently shown that a wrapping interaction between the excluded single-strand of DNA and the outer surface of the helicase is crucial for the unwinding activity of the 3′-5’ MCM helicase from Sulfolobus solfataricus (Sso). Using single-molecule FRET (smFRET), we can now show that this interaction also exists for the hexameric E.coli DnaB (EcDnaB) helicase, which has 5′-3’ polarity, and that similar dynamics are exhibited by both helicases. This suggests that the interaction may be an important component of hexameric helicase unwinding across various helicase superfamilies independent of polarity.We have also investigated the interaction between EcDnaB's inner channel and the encircled-strand using smFRET. A compaction of the encircled strand by the helicase has been suggested based on several crystal structures of hexameric helicases bound to single-strand DNA that exhibits a significant decrease in rise per base. Using EcDnaB, we show that the helicase's binding induces a ‘scrunching’ of the DNA, and that in the absence of ATP, this is a stable interaction. Both excluded-strand wrapping and encircled-strand scrunching are likely critical aspects of replicative helicase unwinding.

Replication of DNA requires helicase and primase activities as part of a primosome assembly. In bacteriophage T4, helicase and primase are separate polypeptides for which little structural information is available and whose mechanism of association within the primosome is not yet understood. Three-dimensional structural information is provided here by means of reconstructions from electron microscopic images. Structures have been calculated for complexes of each of these proteins with ssDNA in the presence of MgATPγS. Both the helicase (gp41) and primase (gp61) complexes are asymmetric hexagonal rings. The gp41 structure suggests two distinct forms that have been termed “open” and “closed.” The gp61 structure is clearly a six-membered ring, which may be a trimer of dimers or a traditional hexamer of monomers. This structure provides conclusive evidence for an oligomeric primase-to-ssDNA stoichiometry of 6:1.

Within replisomes for DNA replication, the primosome is responsible for unwinding double-stranded DNA and synthesizing RNA primers. Assembly of the bacteriophage T4 primosome on individual molecules of ssDNA or forked DNA (fDNA) has been studied by using FRET microscopy. On either DNA substrate, an ordered process of assembly begins with tight 1:1 binding of ssDNA-binding protein (gp32) and helicase-loading protein (gp59) to the DNA. Magnesium adenosine 5′- O -(3-thiotriphosphate) (MgATPγS) mediates the weak binding of helicase (gp41) to DNA coated with gp32 and gp59, whereas MgATP induces gp32 and gp59 to dissociate, leaving gp41 bound to the DNA. Finally, primase (gp61) binds to the gp41·DNA complex. Ensemble studies were used to determine protein stoichiometries and binding constants. These single-molecule studies provide an unambiguous description of the pathway for assembly of the primosome on the lagging strand of DNA at a replication fork.

Structures of the clamp loader–clamp complex reveal that nature uses machined parts in the form of a spiral scaffold (the clamp loader) to thread a circlet (the clamp) on a helix of DNA. The scaffold is then replaced by an interpretive copying machine, the polymerase, to eventually generate the replisome for DNA replication.