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The chemical structure of DNA is fundamental for proper development and function of an organism’s cells and is responsible for the mechanisms of genetic inheritance and evolution. However, the chemical properties of the nucleobases make them highly susceptible to chemical modification and structural alterations that threaten genomic integrity. At the forefront of the battle against such chemical modifications are the DNA glycosylases that initiate the base excision repair pathway (BER) by cleaving erroneous nucleobases from the DNA backbone. The NEIL family of DNA glycosylases (NEIL1, 2, and 3) are unusual from other BER glycosylases in the fact that they can recognize and excise a wide range of modified nucleobases and can do so from several non-canonical DNA contexts. The loss or dysfunction of these enzymes is linked to a wide variety of diseases including metabolic and mental acuity diseases, as well as several types of cancer. Yet the direct relation between these diseases and NEIL activity is unclear. A molecular understanding of the mechanisms of substrate recognition and catalysis will be critical to better understanding their role in cellular disease pathways and to providing potential targets for therapeutic design. Using a variety of chemical biology approaches, this dissertation investigates the mechanisms of NEIL activity on a variety of substrates in multiple DNA contexts and conditions to better understand some of the unusual aspects of NEIL behavior. In chapter two, the ability of NEIL1 and NEIL3 to remove oxidative modifications from G4 DNA in comparison to single strand DNA and canonical duplex DNA contexts was evaluated. Initial studies investigated the ability of the NEIL enzymes to remove Gh from several positions within three potentially G4 forming promoter sequences. By using in vitro glycosylase assays, we observed product production curves that were sequence and enzyme dependent, and that demonstrated base removal by two distinct rates previ

DNA glycosylase, as one member of DNA repair machineries, plays an essential role in correcting mismatched/damaged DNA nucleotides by cleaving the N-glycosidic bond between the sugar and target nucleobase through the base excision repair (BER) pathways. Efficient corrections of these DNA lesions are critical for maintaining genome integrity and preventing premature aging and cancers. The target-site searching/recognition mechanisms and the subsequent conformational dynamics of DNA glycosylase, however, remain challenging to be characterized using experimental techniques. In this review, we summarize our recent studies of sequential structural changes of thymine DNA glycosylase (TDG) during the DNA repair process, achieved mostly by molecular dynamics (MD) simulations. Computational simulations allow us to reveal atomic-level structural dynamics of TDG as it approaches the target-site, and pinpoint the key structural elements responsible for regulating the translocation of TDG along DNA. Subsequently, upon locating the lesions, TDG adopts a base-flipping mechanism to extrude the mispaired nucleobase into the enzyme active-site. The constructed kinetic network model elucidates six metastable states during the base-extrusion process and suggests an active role of TDG in flipping the intrahelical nucleobase. Finally, the molecular mechanism of product release dynamics after catalysis is also summarized. Taken together, we highlight to what extent the computational simulations advance our knowledge and understanding of the molecular mechanism underlying the conformational dynamics of TDG, as well as the limitations of current theoretical work.

The DNA nucleobases are highly susceptible to modification from a wide variety of oxidizing and alkylating agents. Alterations to the nucleobase can threaten genomic integrity by disrupting normal cellular processes including replication and transcription or by introducing mutations. Damage to the DNA base is identified and removed by DNA glycosylases that initiate the base excision repair pathway (BER). The diversity of DNA damage, the location of damage, and how that DNA damage is identified has given valuable insights into disease related to DNA repair. The NEIL family of DNA glycosylases can excise lesions arising from all four nucleobases in a variety of DNA contexts, including duplex, single stranded, and G quadruplex (G4). The loss or dysfunction of NEIL enzymes is associated with a diverse set of disease phenotypes, such as immune deficiencies, anxiety, impaired memory retention, and cancer. Yet, it remains unclear how NEIL dysfunction is related to these phenotypes. In this work, I evaluate the molecular and structural features involved in recognition and excision of a diverse set of lesions across multiple DNA contexts by the NEIL family of glycosylases. First, I examine the features of the damaged nucleobase that influence differences in excision between the two isoforms of NEIL1. RNA editing of the NEIL1 pre-mRNA by the Adenosine Deaminase Acting on RNA (ADAR1) creates two isoforms of NEIL1 via a recoding event that converts a lysine to arginine in the lesion recognition loop of NEIL1. Notably, previous studies have demonstrated that the two isoforms display different enzymatic properties on the pyrimidine lesion, thymine glycol (Tg), where the unedited (UE, K242) isoform showed a significantly faster rate of excision compared to edited NEIL1 (Ed, R242). To further the understanding of NEIL1 activity, I continued this analysis on lesion processing by the two NEIL1 isoforms on a large number of substrates to evaluate the impact of the ADAR1-mediated recod

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2'-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.

The Lipid A moiety of the lipopolysaccharide can be covalently modified during its transport to the outer membrane by different enzymes, among which the LpxT inner membrane protein. LpxT transfers a phosphate group from the undecaprenyl pyrophosphate to the Lipid A, a modification affecting the stability of the outer membrane and its recognition by the host immune system in Enterobacteria. We previously found that the expression of the Pseudomonas aeruginosa lpxT gene, encoding LpxT, is induced in response to a temperature upshift and we proposed that an RNA thermometer was responsible for such regulation. Here we show that the Escherichia coli lpxT orthologous gene is down-regulated upon a temperature upshift and investigated the mechanism of this regulation. We found that the LpxT protein stability is not affected by the temperature change. Conversely, the lpxT mRNA levels strongly decrease upon a shift from 28 to 42 °C. The lack of MicA sRNA, which was previously implicated in lpxT regulation, does not affect lpxT thermal regulation. We identified the lpxTp promoter and demonstrated that lpxTp has temperature-sensitive activity depending on its peculiar −10 region. Moreover, we found that RNase E-dependent degradation of the lpxT mRNA is also modulated by temperature causing a strong destabilization of the lpxT mRNA at 42 °C. In vitro data argue against the involvement of factors differentially expressed at 28 and 42 °C in the temperature–dependent modulation of lpxT mRNA stability.

DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.

MUTYH is a base excision repair (BER) enzyme that prevents mutations in DNA associated with 8-oxoguanine (OG) by catalyzing the removal of adenine from inappropriately formed OG:A base-pairs. Germline mutations in the MUTYH gene are linked to colorectal polyposis and a high risk of colorectal cancer, a syndrome referred to as MUTYH-associated polyposis (MAP). There are over 300 different MUTYH mutations associated with MAP and a large fraction of these gene changes code for missense MUTYH variants. Herein, the adenine glycosylase activity, mismatch recognition properties, and interaction with relevant protein partners of human MUTYH and five MAP variants (R295C, P281L, Q324H, P502L, and R520Q) were examined. P281L MUTYH was found to be severely compromised both in DNA binding and base excision activity, consistent with the location of this variation in the iron-sulfur cluster (FCL) DNA binding motif of MUTYH. Both R295C and R520Q MUTYH were found to have low fractions of active enzyme, compromised affinity for damaged DNA, and reduced rates for adenine excision. In contrast, both Q324H and P502L MUTYH function relatively similarly to WT MUTYH in both binding and glycosylase assays. However, P502L and R520Q exhibited reduced affinity for PCNA (proliferation cell nuclear antigen), consistent with their location in the PCNA-binding motif of MUTYH. Whereas, only Q324H, and not R295C, was found to have reduced affinity for Hus1 of the Rad9-Hus1-Rad1 complex, despite both being localized to the same region implicated for interaction with Hus1. These results underscore the diversity of functional consequences due to MUTYH variants that may impact the progression of MAP.

Among the DNA viruses, the so-called nucleo-cytoplasmic large DNA viruses (NCLDV) constitute a monophyletic group that currently consists of seven families of viruses infecting a very broad variety of eukaryotes, from unicellular marine protists to humans. Many recent papers have analyzed the sequence and structure of NCLDV genomes and their phylogeny, providing detailed analysis about their genomic structure and evolutionary history and proposing their inclusion in a new viral order named Megavirales that, according to some authors, should be considered as a fourth domain of life, aside from Bacteria, Archaea and Eukarya. The maintenance of genetic information protected from environmental attacks and mutations is essential not only for the survival of cellular organisms but also viruses. In cellular organisms, damaged DNA bases are removed in two major repair pathways: base excision repair (BER) and nucleotide incision repair (NIR) that constitute the major pathways responsible for repairing most endogenous base lesions and abnormal bases in the genome by precise repair procedures. Like cells, many NCLDV encode proteins that might constitute viral DNA repair pathways that would remove damages through BER/NIR pathways. However, the molecular mechanisms and, specially, the biological roles of those viral repair pathways have not been deeply addressed in the literature so far. In this paper, we review viral-encoded BER proteins and the genetic and biochemical data available about them. We propose and discuss probable viral-encoded DNA repair mechanisms and pathways, as compared with the functional and molecular features of known homologs proteins.

8-Oxo-7,8-dihydroguanine (8-oxo-Gua, also known as 8-hydroxyguanine) is a major base lesion that is generated by reactive oxygen species in both the DNA and nucleotide pool. The role of DNA glycosylases, which initiate base excision repair, in the mutagenic processes of 8-oxo-Gua in DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP, also known as 8-hydroxy-2'-deoxyguanosine 5'-triphosphate) were investigated using supF shuttle plasmids propagated in human cells. The DNA glycosylases, OGG1, MUTYH, NTH1, and NEIL1, in 293T cells were individually knocked-down by siRNAs and plasmid DNAs containing an 8-oxo-Gua:C/8-oxo-Gua:A pair, and 8-oxo-dGTP plus unmodified plasmid DNA were then introduced into the knocked-down cells. The knock-down of OGG1, MUTYH, NTH1, and NEIL1 resulted in a significant increase in G:C → T:A transversions caused by the 8-oxo-Gua:C pair in the shuttle plasmid. The knock-down of MUTYH resulted in a reduction in A:T → C:G transversions induced by 8-oxo-dGTP and the 8-oxo-Gua:A pair, but the knockdown of OGG1, NTH1, and NEIL1 had no effect on mutagenesis. These results indicate that all of the above DNA glycosylases suppress mutations caused by 8-oxo-Gua:C in DNA. In contrast, it appears that MUTYH enhances A:T → C:G mutations caused by 8-oxo-dGTP.

8-Oxo-7,8-dihydroguanine (8-oxo-Gua, also known as 8-hydroxyguanine) is a major base lesion that is generated by reactive oxygen species in both the DNA and nucleotide pool. The role of DNA glycosylases, which initiate base excision repair, in the mutagenic processes of 8-oxo-Gua in DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP, also known as 8-hydroxy-2'-deoxyguanosine 5'-triphosphate) were investigated using supF shuttle plasmids propagated in human cells. The DNA glycosylases, OGG1, MUTYH, NTH1, and NEIL1, in 293T cells were individually knocked-down by siRNAs and plasmid DNAs containing an 8-oxo-Gua:C/8-oxo-Gua:A pair, and 8-oxo-dGTP plus unmodified plasmid DNA were then introduced into the knocked-down cells. The knock-down of OGG1, MUTYH, NTH1, and NEIL1 resulted in a significant increase in G:C → T:A transversions caused by the 8-oxo-Gua:C pair in the shuttle plasmid. The knock-down of MUTYH resulted in a reduction in A:T → C:G transversions induced by 8-oxo-dGTP and the 8-oxo-Gua:A pair, but the knockdown of OGG1, NTH1, and NEIL1 had no effect on mutagenesis. These results indicate that all of the above DNA glycosylases suppress mutations caused by 8-oxo-Gua:C in DNA. In contrast, it appears that MUTYH enhances A:T → C:G mutations caused by 8-oxo-dGTP.

8-Oxo-7,8-dihydroguanine (8-oxo-Gua, also known as 8-hydroxyguanine) is a major base lesion that is generated by reactive oxygen species in both the DNA and nucleotide pool. The role of DNA glycosylases, which initiate base excision repair, in the mutagenic processes of 8-oxo-Gua in DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP, also known as 8-hydroxy-2'-deoxyguanosine 5'-triphosphate) were investigated using supF shuttle plasmids propagated in human cells. The DNA glycosylases, OGG1, MUTYH, NTH1, and NEIL1, in 293T cells were individually knocked-down by siRNAs and plasmid DNAs containing an 8-oxo-Gua:C/8-oxo-Gua:A pair, and 8-oxo-dGTP plus unmodified plasmid DNA were then introduced into the knocked-down cells. The knock-down of OGG1, MUTYH, NTH1, and NEIL1 resulted in a significant increase in G:C → T:A transversions caused by the 8-oxo-Gua:C pair in the shuttle plasmid. The knock-down of MUTYH resulted in a reduction in A:T → C:G transversions induced by 8-oxo-dGTP and the 8-oxo-Gua:A pair, but the knockdown of OGG1, NTH1, and NEIL1 had no effect on mutagenesis. These results indicate that all of the above DNA glycosylases suppress mutations caused by 8-oxo-Gua:C in DNA. In contrast, it appears that MUTYH enhances A:T → C:G mutations caused by 8-oxo-dGTP.

Human 3-methyladenine-DNA glycosylase (MPG protein) is involved in the base excision repair (BER) pathway responsible mainly for the repair of small DNA base modifications. It initiates BER by recognizing DNA adducts and cleaving the glycosylic bond leaving an abasic site. Here, we explore several of the factors that could influence excision of adducts recognized by MPG, including sequence context, effect of APE1, and interaction with other proteins. To investigate sequence context, we used 13 different 25 bp oligodeoxyribonucleotides containing a unique hypoxanthine residue (Hx) and show that the steady-state specificity of Hx excision by MPG varied by 17-fold. If APE1 protein is used in the reaction for Hx removal by MPG, the steady-state kinetic parameters increase by between fivefold and 27-fold, depending on the oligodeoxyribonucleotide. Since MPG has a role in removing adducts such as 3-methyladenine that block DNA synthesis and there is a potential sequence for proliferating cell nuclear antigen (PCNA) interaction, we hypothesized that MPG protein could interact with PCNA, a protein involved in repair and replication. We demonstrate that PCNA associates with MPG using immunoprecipitation with either purified proteins or whole cell extracts. Moreover, PCNA binds to both APE1 and MPG at different sites, and loading PCNA onto a nicked, closed circular substrate with a unique Hx residue enhances MPG catalyzed excision. These data are consistent with an interaction that facilitates repair by MPG or APE1 by association with PCNA. Thus, PCNA could have a role in short-patch BER as well as in long-patch BER. Overall, the data reported here show how multiple factors contribute to the activity of MPG in cells.

Base excision repair rates of dimethyl sulfate-induced 3-methyladenine and 7-methylguanine adducts were measured at nucleotide resolution along the PGK1 gene in normal human fibroblasts. Rates of 7-methylguanine repair showed a 30-fold dependence on nucleotide position, while position-dependent repair rates of 3-methyladenine varied only sixfold. Slow excision rates for 7-methylguanine bases afforded the opportunity to study their excision in vitro as a model for base excision repair. A two-component in vitro excision system, composed of human N-methylpurine-DNA glycosylase (MPG protein) and dimethyl sulfate-damaged DNA manifested sequence context-dependent rate differences for 7-methylguanine of up to 185-fold from position to position. This in vitro system reproduced both the global repair rate, and for the PGK1 coding region, the position-dependent repair patterns observed in cells. The equivalence of in vivo repair and in vitro excision data indicates that removal of 7-methylguanine by the MPG protein is the rate-limiting step in base excision repair of this lesion. DNA "repair rate footprints" associated with DNA glycosylase accessibility were observed only in a region with bound transcription factors. The "repair rate footprints" represent a rare chromatin component of 7-meG base excision repair otherwise dominated by sequence-context dependence. Comparison of in vivo repair rates to in vitro rates for 3-methyladenine, however, shows that the rate-limiting step determining position-dependent repair for this adduct is at one of the post-DNA glycosylase stages. In conclusion, this study demonstrates that a comparison of sequence context-dependent in vitro reaction rates to in vivo position-dependent repair rates permits the identification of steps responsible for position-dependent repair. Such analysis is now feasible for the different steps and adducts repaired via the base excision repair pathway.