Bypass Polymerases Research Articles

T7 RNA polymerase catalyzed transcription of the epimerizable DNA lesion, Fapy•dG and 8-oxo-2′-deoxyguanosine

Fapy•dG (N6-(2-deoxy-α,β-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamidopyrimidine) and 8-OxodGuo (8-oxo-7,8-dihydro-2′-deoxyguanosine) are major products of 2’-deoxyguanosine oxidation. Fapy•dG is unusual in that it exists as a dynamic mixture of anomers. Much less is known about the effects of Fapy•dG than 8-OxodGuo on transcriptional bypass. The data presented here indicate that T7 RNA polymerase (T7 RNAP) bypass of Fapy•dG is more complex than that of 8-OxodGuo. Primer dependent transcriptional bypass of Fapy•dG by T7 RNAP is hindered compared to dG. T7 RNAP incorporates cytidine opposite Fapy•dG in a miniscaffold at least 13-fold more rapidly than A, G or U. Fitting of reaction data indicate that Fapy•dG anomers are kinetically distinguishable. Extension of a nascent transcript past Fapy•dG is weakly dependent on the nucleotide opposite the lesion. The rate constants describing extension past fast or slow reacting base pairs vary less than 2-fold as a function of the nucleotide opposite the lesion. Promoter dependent T7 RNAP bypass of Fapy•dG and 8-OxodGuo was carried out side-by-side. 8-OxodGuo bypass results in >55% A opposite it. When the shuttle vector contains a Fapy•dG:dA base pair, as high as 20% point mutations and 9% single-nucleotide deletions are produced upon Fapy•dG bypass. Error-prone bypass of a Fapy•dG:dC base pair accounts for ∼9% of the transcripts. Transcriptional bypass mutation frequencies of Fapy•dG and 8-OxodGuo measured in RNA products are comparable to or greater than replication errors, suggesting that these lesions could contribute to mutations significantly through transcription.

Repair and DNA Polymerase Bypass of Clickable Pyrimidine Nucleotides.

Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for bionanotechology applications. Although clickable dNTPs are easily incorporated by DNA polymerases into the growing chain, afterwards they might become targets for DNA repair systems or interfere with faithful nucleotide insertion. Little is known about the possibility and mechanisms of these post-synthetic events. Here, we investigated the repair and (mis)coding properties of EtU and two bulkier clickable pyrimidine nucleosides, 5-(octa-1,7-diyn-1-yl)-U (C8-AlkU) and 5-(octa-1,7-diyn-1-yl)-C (C8-AlkC). In vitro, EtU and C8-AlkU, but not C8-AlkC, were excised by SMUG1 and MBD4, two DNA glycosylases from the base excision repair pathway. However, when placed into a plasmid encoding a fluorescent reporter inactivated by repair in human cells, EtU and C8-AlkU persisted for much longer than uracil or its poorly repairable phosphorothioate-flanked derivative. DNA polymerases from four different structural families preferentially bypassed EtU, C8-AlkU and C8-AlkC in an error-free manner, but a certain degree of misincorporation was also observed, especially evident for DNA polymerase β. Overall, clickable pyrimidine nucleotides could undergo repair and be a source of mutations, but the frequency of such events in the cell is unlikely to be considerable.

Dynamics and thermal stability of the bypass polymerase, DinB homolog (Dbh).

The DinB homolog polymerase (Dbh) is a member of the Y-family of translesion DNA polymerases that can synthesize using a damaged DNA template. Since Dbh comes from the thermophilic archaeon Sulfolobus acidocaldarius, it is capable of functioning over a wide range of temperatures. Existing X-ray structures were determined at temperatures where the protein is least active. Here we use NMR and circular dichroism to understand how the structure and dynamics of Dbh are affected by temperature (2°C-65°C) and metal ion binding in solution. We measured hydrogen exchange protection factors, temperature coefficients, and chemical shift perturbations with and without magnesium and manganese. We report on regions of the protein that become more dynamic as the temperature is increased toward the functional temperature. Hydrogen exchange protection factors and temperature coefficients reveal that both the thumb and finger domains are very dynamic relative to the palm and little-finger (LF) domains. These trends remain true at high temperature with dynamics increasing as temperatures increase from 35°C to 50°C. Notably, NMR spectra show that the Dbh tertiary structure cold denatures beginning at 25°C and increases in denaturation as the temperature is lowered to 5°C with little change observed by CD. Above 35°C, chemical shift perturbation analysis in the presence and absence of magnesium and manganese reveals three ion binding sites, without DNA bound. In contrast, these bound metals are not apparent in any Dbh crystal structures of the protein without DNA. Two ion binding sites are confirmed to be near the active site, as reported in other Y-family polymerases, and we report a novel ion binding site in the LF domain. Thus, the solution-state structure of the Dbh polymerase is distinct from that of the solid-state structures and shows an unusually high cold denaturation temperature.

NEIL3 promoter G-quadruplex with oxidatively modified bases shows magnesium-dependent folding that stalls polymerase bypass

Oxidative stress unleashes reactive species capable of oxidizing 2′-deoxyguanosine (G) nucleotides in G-rich sequences of the genome, such as the potential G-quadruplex forming sequencing (PQS) in the NEIL3 gene promoter. Oxidative modification of G yields 8-oxo-7,8-dihydro-2′-deoxyguanosine (OG) that can be further oxidized to hydantoin products. Herein, OG was synthesized into the NEIL3 PQS that was allowed to fold to a G-quadruplex (G4) in K+ ion solutions with varying amounts of Mg2+ in the physiological range. The Mg2+ dependency in the oxidatively modified NEIL3 G4 to stall a replicative DNA polymerase was evaluated. The polymerase was found to stall at the G4 or OG, as well as continue to full-length extension with dependency on the location of the modification and the concentration of Mg2+. To provide some clarity on these findings, OG or the hydantoins were synthesized in model NEIL3 G4 folding sequences at the positions of the polymerase study. The model G4 sequences were allowed to fold in K+ ion solutions with varying levels of Mg2+ to identify how the presence of the divalent metal impacted G4 folding depending on the location of the modification. The presence of Mg2+ either caused the transition of the NEIL3 G4 folds from an antiparallel to parallel orientation of the strands or had no impact. Structural models are proposed to understand the findings using the literature as a guide. The biological significance of the results is discussed.

Dynamics of the bypass polymerase, dinB homolog.

The dinB homolog (Dbh) polymerase is a member of the Y-family of translesion DNA polymerases, able to synthesize DNA across damaged DNA template. Since Dbh is derived from the thermophilic archaeon Sulfolobus acidocaldarius, it is capable of functioning at high temperatures where mesophilic proteins would be denatured. Here we use NMR and Circular Dichroism (CD) to compare protein stability at a wide range of temperatures. 1H-15N HSQC spectra show a change of tertiary structures starting at 25 °C and progression at colder temperatures (15, 5 °C). CD show retentions of secondary structure with a slight decrease percent helicity and an increase in percent beta strand as temperature decrease. We have analyzed hydrogen exchange measurement at 35°C and 50°C to investigate the folding dynamics of Dbh. We observe the moderate modulation of dynamics upon heating. Core residues in the palm and little finger domain, especially those in the interior of the β-sheets and interior face of α-helices, are key in maintaining the stability of Dbh; with the palm providing a stable platform for the active site residues. We report on regions of the protein that become more dynamic as the temperature is increased toward the functional temperature.

UVA Radiation, DNA Damage, and Melanoma.

Melanoma is a lethal type of skin tumor that has been linked with sunlight exposure chiefly in fair-skinned human populations. Wavelengths from the sun that can reach the earth’s surface include UVA radiation (320–400 nm) and UVB radiation (280–320 nm). UVB effectively induces the formation of dimeric DNA photoproducts, preferentially the cyclobutane pyrimidine dimers (CPDs). The characteristic UVB signature mutations in the form of C to T mutations at dipyrimidine sequences are prevalent in melanoma tumor genomes and have been ascribed to deamination of cytosines within CPDs before DNA polymerase bypass. However, evidence from epidemiological, animal, and other experimental studies also suggest that UVA radiation may participate in melanoma formation. The DNA damage relevant for UVA includes specific types of CPDs at TT sequences and perhaps oxidative DNA damage to guanine, both induced by direct or indirect, photosensitization-mediated chemical and biophysical processes. We summarize the evidence for a potential role of UVA in melanoma and discuss some of the mechanistic pathways of how UVA may induce mutagenesis in melanocytes.

Site-Specific 5-Formyl Cytosine Mediated DNA-Histone Cross-Links: Synthesis and Polymerase Bypass by Human DNA Polymerase η.

DNA-protein cross-links (DPCs) between DNA epigenetic mark 5-formylC and lysine residues of histone proteins spontaneously form in human cells. Such conjugates are likely to influence chromatin structure and mediate DNA replication, transcription, and repair, but are challenging to study due to their reversible nature. Here we report the construction of site specific, hydrolytically stable DPCs between 5fdC in DNA and K4 of histone H3 and an investigation of their effects on DNA replication. Our approach employs oxime ligation, allowing for site-specific conjugation of histones to DNA under physiological conditions. Primer extension experiments revealed that histone H3-DNA crosslinks blocked DNA synthesis by hPol η polymerase, but were bypassed following proteolytic processing.

Site‐Specific 5‐Formyl Cytosine Mediated DNA‐Histone Cross‐Links: Synthesis and Polymerase Bypass by Human DNA Polymerase η

AbstractDNA‐protein cross‐links (DPCs) between DNA epigenetic mark 5‐formylC and lysine residues of histone proteins spontaneously form in human cells. Such conjugates are likely to influence chromatin structure and mediate DNA replication, transcription, and repair, but are challenging to study due to their reversible nature. Here we report the construction of site specific, hydrolytically stable DPCs between 5fdC in DNA and K4 of histone H3 and an investigation of their effects on DNA replication. Our approach employs oxime ligation, allowing for site‐specific conjugation of histones to DNA under physiological conditions. Primer extension experiments revealed that histone H3‐DNA crosslinks blocked DNA synthesis by hPol η polymerase, but were bypassed following proteolytic processing.

Translesion Synthesis Past 5-Formylcytosine-Mediated DNA-Peptide Cross-Links by hPolη Is Dependent on the Local DNA Sequence.

DNA-protein cross-links (DPCs) are unusually bulky DNA lesions that form when cellular proteins become trapped on DNA following exposure to ultraviolet light, free radicals, aldehydes, and transition metals. DPCs can also form endogenously when naturally occurring epigenetic marks [5-formyl cytosine (5fC)] in DNA react with lysine and arginine residues of histones to form Schiff base conjugates. Our previous studies revealed that DPCs inhibit DNA replication and transcription but can undergo proteolytic cleavage to produce smaller DNA-peptide conjugates. We have shown that 5fC-conjugated DNA-peptide cross-links (DpCs) placed within the CXA sequence (X = DpC) can be bypassed by human translesion synthesis (TLS) polymerases η and κ in an error-prone manner. However, the local nucleotide sequence context can have a strong effect on replication bypass of bulky lesions by influencing the geometry of the ternary complex among the DNA template, polymerase, and the incoming dNTP. In this work, we investigated polymerase bypass of 5fC-DNA-11-mer peptide cross-links placed in seven different sequence contexts (CXC, CXG, CXT, CXA, AXA, GXA, and TXA) in the presence of human TLS polymerase η. Primer extension products were analyzed by gel electrophoresis, and steady-state kinetics of the misincorporation of dAMP opposite the DpC lesion in different base sequence contexts was investigated. Our results revealed a strong impact of nearest neighbor base identity on polymerase η activity in the absence and presence of a DpC lesion. Molecular dynamics simulations were used to structurally explain the experimental findings. Our results suggest a possible role of local DNA sequence in promoting TLS-related mutational hot spots in the presence and absence of DpC lesions.

Synthesis and polymerase bypass studies of DNA-peptide and DNA-protein conjugates.

DNA-peptide (DpCs) and DNA-protein cross-links (DPCs) are DNA lesions formed when polypeptides and nuclear proteins become covalently trapped on DNA strands. DNA-protein cross-links are of enormous size and hence pose challenges to cell survival by blocking DNA replication, transcription, and repair. However, DPCs can undergo proteolytic degradation via various pathways to give shorter polypeptide chains (DpCs). The resulting DpC lesions are efficiently bypassed by translesion synthesis (TLS) DNA polymerases like κ, η, δ, etc., although polymerase bypass efficiency as well as correct base insertion depends heavily on size, sequence context, and position of peptides in DpCs. This chapter explores various synthetic methods to generate these lesions including detailed experimental procedures for the construction of DpCs and DPCs via reductive amination and oxime ligation. Further we describe biochemical experiments to investigate the effects of these lesions on DNA polymerase activity and fidelity.

Structural biology of DNA abasic site protection by SRAP proteins

Abasic (AP) sites are one of the most frequently occurring types of DNA damage. They lead to DNA strand breaks, interstrand DNA crosslinks, and block transcription and replication. Mutagenicity of AP sites arises from translesion synthesis (TLS) by error-prone bypass polymerases. Recently, a new cellular response to AP sites was discovered, in which the protein HMCES (5-hydroxymethlycytosine (5hmC) binding, embryonic stem cell-specific) forms a stable, covalent DNA-protein crosslink (DPC) to AP sites at stalled replication forks. The stability of the HMCES-DPC prevents strand cleavage by endonucleases and mutagenic bypass by TLS polymerases. Crosslinking is carried out by a unique SRAP (SOS Response Associated Peptidase) domain conserved across all domains of life. Here, we review the collection of recently reported SRAP crystal structures from human HMCES and E. coli YedK, which provide a unified basis for SRAP specificity and a putative chemical mechanism of AP site crosslinking. We discuss the structural and chemical basis for the stability of the SRAP DPC and how it differs from covalent protein-DNA intermediates in DNA lyase catalysis of strand scission.

Genetic Control of Oxidative Mutagenesis in Sulfolobus acidocaldarius.

To identify DNA-oxidation defenses of hyperthermophilic archaea, we deleted genes encoding the putative 7,8-dihydro-8-oxoguanine (oxoG)-targeted N-glycosylase of S. acidocaldarius (ogg; Saci_01367), the Y-family DNA polymerase (dbh; Saci_0554), or both, and measured the effects on cellular survival, replication accuracy, and oxoG bypass in vivo Spontaneous G:C to T:A transversions were elevated in all Δogg and Δdbh constructs, and the Δogg Δdbh double mutant lost viability at a faster rate than isogenic WT and ogg strains. The distribution of G:C to T:A transversions within mutation-detector genes suggested that reactivity of G toward oxidation and the effect on translation contribute heavily to the pattern of mutations that are recovered. An impact of the Ogg protein on overall efficiency of bypassing oxoG in transforming DNA was evident only in the absence of Dbh, and Ogg status did not affect the accuracy of bypass. Dbh function, in contrast, dramatically influenced both the efficiency and accuracy of oxoG bypass. Thus, Ogg and Dbh were found to work independently to avoid mutagenesis by oxoG, and inactivating this simple but effective defense system by deleting both genes imposed a severe mutational burden on S. acidocaldarius cells.IMPORTANCE Hyperthermophilic archaea are expected to have effective (and perhaps atypical) mechanisms to limit the genetic consequences of DNA damage, but few gene products have been demonstrated to have genome-preserving functions in vivo This study confirmed by genetic criteria that the S. acidocaldarius Ogg protein avoids the characteristic mutagenesis of G oxidation. This enzyme and the bypass polymerase Dbh have similar impacts on genome stability but work independently, and may comprise most of the DNA-oxidation defense of S. acidocaldarius The critical dependence of accurate oxoG bypass on the accessory DNA polymerase Dbh further argues that some form of polymerase exchange is important for accurate genome replication in Sulfolobus, and perhaps in related hyperthermophilic archaea.

On the Enzymatic Formation of Metal Base Pairs with Thiolated and pKa -Perturbed Nucleotides.

The formation of artificial metal base pairs is an alluring and versatile method for the functionalization of nucleic acids. Access to DNA functionalized with metal base pairs is granted mainly by solid-phase synthesis. An alternative, yet underexplored method, envisions the installation of metal base pairs through the polymerization of modified nucleoside triphosphates. Herein, we have explored the possibility of using thiolated and pKa -perturbed nucleotides for the enzymatic construction of artificial metal base pairs. The thiolated nucleotides S2C, S6G, and S4T as well as the fluorinated analogue 5FU are readily incorporated opposite a templating S4T nucleotide through the guidance of metal cations. Multiple incorporation of the modified nucleotides along with polymerase bypass of the unnatural base pairs are also possible under certain conditions. The thiolated nucleotides S4T, S4T, S2C, and S6G were also shown to be compatible with the synthesis of modified, high molecular weight single-stranded (ss)DNA products through TdT-mediated tailing reactions. Thus, sulfur-substitution and pKa perturbation represent alternative strategies for the design of modified nucleotides compatible with the enzymatic construction of metal base pairs.

Adduct Fluorescence as a Tool to Decipher Sequence Impact on Frameshift Mutations Mediated by a C-Linked C8-Biphenyl-Guanine Lesion.

Aromatic chemicals can undergo metabolic activation to afford electrophilic species that react at the C8-site of 2'-deoxyguanosine (dG) to generate bulky C8-dG adducts as a basis of initiating carcinogenesis. These DNA lesions have served as models to understand the mechanism of frameshift mutagenesis, especially within CG-dinucleotide repeat sequences, such as NarI (5'-GGCXCC-3', where X = C8-dG adduct), however there is still limited capacity to predict the likelihood of mutation arising within particular contexts, and hence chemistry-based strategies are needed for probing relationships between nucleic acid sequence and structure with replication errors. In the NarI sequence, certain C8-dG adducts may trigger in the course of DNA synthesis the formation of a slipped mutagenic intermediate (SMI) that contains a two nucleotide (XC) bulge in the template strand that can form upstream of the polymerase active site. This distortion facilitates polymerization but affords a GC dinucleotide deletion product (-2 frameshift mutation). In the current study, incorporating the fluorescent C-linked 4-fluorobiphenyl-dG (FBP-dG) adduct into two 22-mer templates containing CG-dinucleotide repeats ( NarI: 3'-CXCGGC-5' and CG3: 3'-CXCGCG-5', X = FBP-dG) and performing primer extension reactions using DNA polymerase I, Klenow fragment exo- (Kf-) revealed a dramatic sequence-based difference in polymerase bypass efficiency. Primer extension past FBP-dG within the NarI sequence was strongly blocked, whereas Kf- extended the primer past FBP-dG within a CG3 template to afford a full-length product and the GC dinucleotide deletion. To model the nucleotide insertion steps in the fully paired (FP) versus the slipped mutagenic (SM) translesion pathways, adducted template:primer duplexes were constructed and characterized by UV thermal denaturation and fluorescence spectroscopy. The emission intensity of the FBP-dG lesion exhibits sensitivity to SMI formation (turn-on) versus a FP duplex (turn-off), permitting insight into adduct base-pairing within the template:primer duplexes. This fluorescence sensitivity provides a rationale for sequence impact on -2 frameshift mutations mediated by the C-linked FBP-dG lesion.

Conformational Flexibility of the Benzyl-Guanine Adduct in a Bypass Polymerase Active Site Permits Replication: Insights from Molecular Dynamics Simulations.

Although translesion synthesis (TLS) polymerases play key roles in replicating DNA that contains nucleobase addition products (adducts), there are many unknowns about their function. The present work gains indispensable structural insights from molecular dynamics simulations on the replication of O6-benzyl-guanine (Bz-dG) prior to bond formation during dCTP insertion opposite the adduct by Dpo4. When combined with previous X-ray crystal structures of the Bz-dG extension complex, molecular details are now available for each stage during a single TLS replication cycle for this carcinogenic lesion. Our calculations illustrate that Bz-dG preferentially adopts an intercalated bulky moiety orientation in the Dpo4 preinsertion complex, which stabilizes the complex through Bz-dG interactions with the previously replicated 3'-base pair and positions the carcinogenic group in the dNTP binding site. Nevertheless, the maintained inherent flexibility of Bz-dG due to a stark lack of interactions with the polymerase or template DNA allows the bulky moiety to adopt a major groove position opposite an incoming dCTP in an orientation that is conducive for the experimentally observed nonmutagenic bypass. Comparison of Bz-dG and canonical dG replication clarifies that the experimentally observed decrease in dCTP binding affinity and replication efficiency upon adduct formation is likely caused by a combination of factors, including the required template nucleotide conformational change and destabilized template-dCTP hydrogen bonding. Although additional aspects of the replication process, such as the impact of the adduct on the nucleotidyl-transfer reaction, may also be important for fully rationalizing experimental replication data and must be considered in future work, the present contribution emphasizes the importance of considering the effect of DNA damage on the early stages of the TLS process.

Plant organellar DNA polymerases are replicative and translesion DNA synthesis polymerases.

Genomes acquire lesions that can block the replication fork and some lesions must be bypassed to allow survival. The nuclear genome of flowering plants encodes two family-A DNA polymerases (DNAPs), the result of a duplication event, that are the sole DNAPs in plant organelles. These DNAPs, dubbed Plant Organellar Polymerases (POPs), resemble the Klenow fragment of bacterial DNAP I and are not related to metazoan and fungal mitochondrial DNAPs. Herein we report that replicative POPs from the plant model Arabidopsis thaliana (AtPolI) efficiently bypass one the most insidious DNA lesions, an apurinic/apyrimidinic (AP) site. AtPolIs accomplish lesion bypass with high catalytic efficiency during nucleotide insertion and extension. Lesion bypass depends on two unique polymerization domain insertions evolutionarily unrelated to the insertions responsible for lesion bypass by DNAP θ, an analogous lesion bypass polymerase. AtPolIs exhibit an insertion fidelity that ranks between the fidelity of replicative and lesion bypass DNAPs, moderate 3′-5′ exonuclease activity and strong strand-displacement. AtPolIs are the first known example of a family-A DNAP evolved to function in both DNA replication and lesion bypass. The lesion bypass capabilities of POPs may be required to prevent replication fork collapse in plant organelles.

1,3-Butadiene-Induced Adenine DNA Adducts Are Genotoxic but Only Weakly Mutagenic When Replicated in Escherichia coli of Various Repair and Replication Backgrounds.

The adverse effects of the human carcinogen 1,3-butadiene (BD) are believed to be mediated by its DNA-reactive metabolites such as 3,4-epoxybut-1-ene (EB) and 1,2,3,4-diepoxybutane (DEB). The specific DNA adducts responsible for toxic and mutagenic effects of BD, however, have yet to be identified. Recent in vitro polymerase bypass studies of BD-induced adenine (BD-dA) adducts show that DEB-induced N6,N6-DHB-dA (DHB = 2,3-dihydroxybutan-1,4-diyl) and 1,N6-γ-HMHP-dA (HMHP = 2-hydroxy-3-hydroxymethylpropan-1,3-diyl) adducts block replicative DNA polymerases but are bypassed by human polymerases η and κ, leading to point mutations and deletions. In contrast, EB-induced N6-HB-dA (HB = 2-hydroxy-3-buten-1-yl) does not block DNA synthesis and is nonmutagenic. In the present study, we employed a newly established in vivo lesion-induced mutagenesis/genotoxicity assay via next-generation sequencing to evaluate the in vivo biological consequences of S-N6-HB-dA, R,R-N6,N6-DHB-dA, S,S-N6,N6-DHB-dA, and R,S-1,N6-γ-HMHP-dA. In addition, the effects of AlkB-mediated direct reversal repair, MutM and MutY catalyzed base excision repair, and DinB translesion synthesis on the BD-dA adducts in bacterial cells were investigated. BD-dA adducts showed the expected inhibition of DNA replication in vivo but were not substantively mutagenic in any of the genetic environments investigated. This result is in contrast with previous in vitro observations and opens the possibility that E. coli repair and bypass systems other than the ones studied here are able to minimize the mutagenic properties of BD-dA adducts.

Repairing Interstrand DNA Crosslinks (ICL): Characterization of an ICL Incision/Lesion Bypass Polymerase Complex regulated by the Fanconi anemia pathway

The ‘REVersionless’ genes, REV1, REV3, and REV7, were discovered as being necessary for both spontaneous and DNA damage‐induced mutagenesis in yeast. REV3 is the catalytic subunit of DNA polymerase Zeta (ζ) and together with its accessory subunit REV7 extends nucleotides inserted opposite multiple types of DNA lesions. REV1 is a deoxycytidyl transferase and performs a scaffolding function in orchestrating the switch between insertion TLS polymerases directly opposite a DNA adduct and Polζ for the extension step. REV1 and Polζ are also involved in ICL repair and homologous recombination (HR) repair of DNA double stranded breaks (DSB). The phenotypes of cells deficient in expression of REV1, REV3, or REV7 are similar to cells derived from patients diagnosed with Fanconi Anemia (FA), a genetic disorder characterized by bone marrow failure, ICL hypersensitivity, and increased risk for cancer. Previous studies have demonstrated an epistatic relationship between REV1 or REV3 and genes encoding proteins comprising the FA core signaling complex in resistance to cisplatin. Additionally, REV1, Polζ, and the FANCD2/FANCI heterodimer have been shown individually to be involved in homologous recombination (HR) repair, which is crucial for resolution of DNA DSBs and ICL repair during S phase. We demonstrate that human REV1 and REV3 exhibit an epistatic relationship with FANCD2 and FANCI in cellular resistance and ICL repair following mitomycin C (MMC) treatment, as well as HR repair. MMC‐induced chromosomal anomalies are increased in REV1 or REV3‐depleted cells and FANCD2 depletion does not further exacerbate this phenotype. We further show that the FANCA core complex protein regulates MMC‐induced chromatin association of REV7 and by extension REV3 to sites of ICL repair. Co‐immunoprecipitation studies show that REV1, REV3 and REV7 interact with FANCD2 and FANCI independent of DNA damage or monoubiquitination of FANCD2 by the FA core complex. In addition, REV1, REV3, and REV7 co‐immunoprecipitate with the SLX4/FANCP scaffold protein, which regulates the unhooking step of an ICL DNA adduct by the ERCC1/XPF (FANCQ) endonuclease and HR repair of a DSB. We propose a model where REV1/Polζ‐dependent activities during ICL and HR repair are regulated by multiple FA proteins including FANCA, FANCD2 and SLX4 (FANCP). Together, these results suggest that REV1 and Polζ are part of a macromolecular complex involved in resistance to many anticancer agents and interactions within this complex may be viable targets for small molecule inhibitors to sensitize resistant tumors to chemotherapy.Support or Funding InformationR01 CA133046

Advances in Structural and Single-Molecule Methods for Investigating DNA Lesion Bypass and Repair Polymerases.

Innovative advances in X-ray crystallography and single-molecule biophysics have yielded unprecedented insight into the mechanisms of DNA lesion bypass and damage repair. Time-dependent X-ray crystallography has been successfully applied to view the bypass of 8-oxo-7,8-dihydro-2'-deoxyguanine (8-oxoG), a major oxidative DNA lesion, and the incorporation of the triphosphate form, 8-oxo-dGTP, catalyzed by human DNA polymerase β. Significant findings of these studies are highlighted here, and their contributions to the current mechanistic understanding of mutagenic translesion DNA synthesis (TLS) and base excision repair are discussed. In addition, single-molecule Förster resonance energy transfer (smFRET) techniques have recently been adapted to investigate nucleotide binding and incorporation opposite undamaged dG and 8-oxoG by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. The mechanistic response of Dpo4 to a DNA lesion and the complex smFRET technique are described here. In this perspective, we also describe how time-dependent X-ray crystallography and smFRET can be used to achieve the spatial and temporal resolutions necessary to answer some of the mechanistic questions that remain in the fields of TLS and DNA damage repair.

Synthesis of N7-Alkyl-9-deaza-2'-deoxyguanosines Containing Polar N7 Chains. Examples of Chemically Stable Analogues of N7-Hydroxyethyl and N7-Oxoethyl Adducts of 2'-Deoxyguanosine.

Development of chemically stable analogues of unstable DNA lesions enables accurate study of polymerase bypass. We report the design and synthesis of N7-hydroxyethyl-9-deaza-2'-deoxyguanosine and N7-oxoethyl-9-deaza-2'-deoxyguanosine as the analogues of N7-hydroxyethyl-2'-deoxyguanosine and N7-oxoethyl-2'-deoxyguanosine, respectively. We also developed the synthesis of these two nucleosides whose N7 side chains are protected by TBS for the convenience of conversion to phosphoramidites.