Deoxyribose Phosphate Research Articles

Kinetic mechanism of the interaction of Saccharomyces cerevisiae AP-endonuclease 1 with DNA substrates

The apurinic/apyrimidinic endonuclease from Saccharomyces cerevisiae Apn1 is one of the key enzymes involved in base excision repair of DNA lesions. A major function of the enzyme is to cleave the upstream phosphodiester bond of an apurinic/apyrimidinic site (AP-site), leading to the formation of a single-strand break with 3'-hydroxyl (OH) and 5'-deoxyribose phosphate (dRP) termini. In this study, the pre-steady-state kinetics and conformational dynamics of DNA substrates during their interaction with Apn1 were investigated. A stopped-flow method with detection of the fluorescence intensity of 2-aminopurine and pyrrolocytosine located adjacent or opposite to the damage was used. It was found that upon interaction with Apn1, both DNA strands undergo a number of rapid changes. The location of fluorescent analogs of heterocyclic bases in DNA does not influence the catalytic step of the reaction. Comparison of data obtained for yeast Apn1 and reported data (Kanazhevskaya, L. Yu., Koval, V. V., Vorobjev, Yu. N., and Fedorova, O. S. (2012) Biochemistry, 51, 1306-1321) for human Ape1 revealed some differences in their interaction with DNA substrates.

HMGN1 Protein Regulates Poly(ADP-ribose) Polymerase-1 (PARP-1) Self-PARylation in Mouse Fibroblasts

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.

Colon Cancer-associated DNA Polymerase β Variant Induces Genomic Instability and Cellular Transformation

Rapidly advancing technology has resulted in the generation of the genomic sequences of several human tumors. We have identified several mutations of the DNA polymerase β (pol β) gene in human colorectal cancer. We have demonstrated that the expression of the pol β G231D variant increased chromosomal aberrations and induced cellular transformation. The transformed phenotype persisted in the cells even once the expression of G231D was extinguished, suggesting that it resulted as a consequence of genomic instability. Biochemical analysis revealed that its catalytic rate was 140-fold slower than WT pol β, and this was a result of the decreased binding affinity of nucleotides by G231D. Residue 231 of pol β lies in close proximity to the template strand of the DNA. Molecular modeling demonstrated that the change from a small and nonpolar glycine to a negatively charged aspartate resulted in a repulsion between the template and residue 231 leading to the distortion of the dNTP binding pocket. In addition, expression of G231D was insufficient to rescue pol β-deficient cells treated with chemotherapeutic agents suggesting that these agents may be effectively used to treat tumors harboring this mutation. More importantly, this suggests that the G231D variant has impaired base excision repair. Together, these data indicate that the G231D variant plays a role in driving cancer.

Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Repairs DNA Damage Induced by Topoisomerases I and II and Base Alkylation in Vertebrate Cells

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs topoisomerase I cleavage complexes (Top1cc) by hydrolyzing their 3'-phosphotyrosyl DNA bonds and repairs bleomycin-induced DNA damage by hydrolyzing 3'-phosphoglycolates. Yeast Tdp1 has also been implicated in the repair of topoisomerase II-DNA cleavage complexes (Top2cc). To determine whether vertebrate Tdp1 is involved in the repair of various DNA end-blocking lesions, we generated Tdp1 knock-out cells in chicken DT40 cells (Tdp1-/-) and Tdp1-complemented DT40 cells with human TDP1. We found that Tdp1-/- cells were not only hypersensitive to camptothecin and bleomycin but also to etoposide, methyl methanesulfonate (MMS), H(2)O(2), and ionizing radiation. We also show they were deficient in mitochondrial Tdp1 activity. In biochemical assays, recombinant human TDP1 was found to process 5'-phosphotyrosyl DNA ends when they mimic the 5'-overhangs of Top2cc. Tdp1 also processes 3'-deoxyribose phosphates generated from hydrolysis of abasic sites, which is consistent with the hypersensitivity of Tdp1-/- cells to MMS and H(2)O(2). Because recent studies established that CtIP together with BRCA1 also repairs topoisomerase-mediated DNA damage, we generated dual Tdp1-CtIP-deficient DT40 cells. Our results show that Tdp1 and CtIP act in parallel pathways for the repair of Top1cc and MMS-induced lesions but are epistatic for Top2cc. Together, our findings reveal a broad involvement of Tdp1 in DNA repair and clarify the role of human TDP1 in the repair of Top2-induced DNA damage.

Selection of a New Whole Cell Biocatalyst for the Synthesis of 2-Deoxyribose 5-Phosphate

2-deoxyribose 5-phosphate (DR5P) is a key intermediate in the biocatalyzed preparation of deoxyribonucleosides. Therefore, DR5P production by means of simpler, cleaner, and economic pathways becomes highly interesting. One strategy involves the use of bacterial whole cells containing DR5P aldolase as biocatalyst for the aldol addition between acetaldehyde and D: -glyceraldehyde 3-phosphate or glycolytic intermediates that in situ generate the acceptor substrate. In this work, diverse microorganisms capable of synthesizing DR5P were selected by screening several bacteria genera. In particular, Erwinia carotovora ATCC 33260 was identified as a new biocatalyst that afforded 14.1-mM DR5P starting from a cheap raw material like glucose.

The Crystal Structure of Streptococcus pyogenes Uridine Phosphorylase Reveals a Distinct Subfamily of Nucleoside Phosphorylases

Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 Å resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an α/β monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is ∼7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.

Uracil-DNA Glycosylase of Thermoplasma acidophilumDirects Long-Patch Base Excision Repair, Which Is Promoted by Deoxynucleoside Triphosphates and ATP/ADP, into Short-Patch Repair

Hydrolytic deamination of cytosine to uracil in DNA is increased in organisms adapted to high temperatures. Hitherto, the uracil base excision repair (BER) pathway has only been described in two archaeons, the crenarchaeon Pyrobaculum aerophilum and the euryarchaeon Archaeoglobus fulgidus, which are hyperthermophiles and use single-nucleotide replacement. In the former the apurinic/apyrimidinic (AP) site intermediate is removed by the sequential action of a 5'-acting AP endonuclease and a 5'-deoxyribose phosphate lyase, whereas in the latter the AP site is primarily removed by a 3'-acting AP lyase, followed by a 3'-phosphodiesterase. We describe here uracil BER by a cell extract of the thermoacidophilic euryarchaeon Thermoplasma acidophilum, which prefers a similar short-patch repair mode as A. fulgidus. Importantly, T. acidophilumcell extract also efficiently executes ATP/ADP-stimulated long-patch BER in the presence of deoxynucleoside triphosphates, with a repair track of ∼15 nucleotides. Supplementation of recombinant uracil-DNA glycosylase (rTaUDG; ORF Ta0477) increased the formation of short-patch at the expense of long-patch repair intermediates, and additional supplementation of recombinant DNA ligase (rTalig; Ta1148) greatly enhanced repair product formation. TaUDG seems to recruit AP-incising and -excising functions to prepare for rapid single-nucleotide insertion and ligation, thus excluding slower and energy-costly long-patch BER.

Probing the Active Site of the Deoxynucleotide N-Hydrolase Rcl Encoded by the Rat Gene c6orf108

Rcl is a potential anti-angiogenic therapeutic target that hydrolyzes the N-glycosidic bond of 2'-deoxyribonucleoside 5'-monophosphate, yielding 2-deoxyribose 5-phosphate and the corresponding base. Its recently elucidated solution structure provided the first insight into the molecular basis for the substrate recognition. To facilitate the development of potent and specific inhibitors of Rcl, the active site was probed by site-directed mutagenesis and by the use of substrate analogs. The nucleobase shows weak interactions with the protein, and the deoxyribose binding pocket includes the catalytic triad Tyr-13, Asp-69, and Glu-93 and the phosphate binding site Ser-87 and Ser-117. The phosphomimetic mutation of Ser-17 to Glu prevents substrate binding and, thus, abolishes the activity of Rcl. The synthetic ligand-based analysis of the Rcl binding site shows that substitutions at positions 2 and 6 of the nucleobase as well as large heterocycles are well tolerated. The phosphate group at position 5 of the (deoxy)ribose moiety is the critical binding determinant. This study provides the roadmap for the design of small molecules inhibitors with pharmacological properties.

Association between the Herpes Simplex Virus-1 DNA Polymerase and Uracil DNA Glycosylase

Herpes simplex virus-1 (HSV-1) is a large dsDNA virus that encodes its own DNA replication machinery and other enzymes involved in DNA transactions. We recently reported that the HSV-1 DNA polymerase catalytic subunit (UL30) exhibits apurinic/apyrimidinic and 5'-deoxyribose phosphate lyase activities. Moreover, UL30, in conjunction with the viral uracil DNA glycosylase (UL2), cellular apurinic/apyrimidinic endonuclease, and DNA ligase IIIalpha-XRCC1, performs uracil-initiated base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we show that the HSV-1 UL2 associates with the viral replisome. We identified UL2 as a protein that co-purifies with the DNA polymerase through numerous chromatographic steps, an interaction that was verified by co-immunoprecipitation and direct binding studies. The interaction between UL2 and the DNA polymerase is mediated through the UL30 subunit. Moreover, UL2 co-localizes with UL30 to nuclear viral prereplicative sites. The functional consequence of this interaction is that replication of uracil-containing templates stalls at positions -1 and -2 relative to the template uracil because of the fact that these are converted into non-instructional abasic sites. These findings support the existence of a viral repair complex that may be capable of replication-coupled base excision repair and further highlight the role of DNA repair in the maintenance of the HSV-1 genome.

Production of 2-deoxyribose 5-phosphate from fructose to demonstrate a potential of artificial bio-synthetic pathway using thermophilic enzymes

Six thermophilic enzymes from Thermus thermophilus were used to construct an ‘artificial bio-synthetic pathway’ for the production of 2-deoxyribose 5-phosphate from fructose. By a simple operation using six recombinant Escherichia coli strains producing the thermophilic enzymes, respectively, fructose was converted to 2-deoxyribose 5-phosphate with a molar yield of 55%.

Expression and Characterization of a Novel Deoxyribose 5-Phosphate Aldolase from Paenibacillus sp. EA001

A novel deoC gene was identified from Paenibacillus sp. EA001 isolated from soil. The gene had an open reading frame (ORF) of 663 base pairs encoding 220 amino acids with a molecular mass of 24.5 kDa. The amino acid sequence was 79 % identical to that of deoxyribose 5-phosphate aldolase (DERA) from Geobacillus sp. Y412MC10. The deoC gene encoding DERA was cloned into expression vector and the protein was expressed in Escherichia coli. The recombinant DERA was purified by using Ni-NTA affinity chromatography and characterized. The optimum temperature and pH for DERA were 50 degrees C and 6.0, respectively. The specific activity for deoxyribose 5-phosphate (DR5P), substrate, was 62 micronmol/min/mg. The Km value for DR5P was determined to be 145 mM with the Kcat value of 3.2 times 10(2 /sec) from Lineweaver-Burk plots. The EA001 DERA showed stability toward a high concentration of acetaldehyde (100 mM).

Toxoplasma gondii deoxyribose phosphate aldolase-like protein ( TgDPA) interacts with actin depolymerizing factor ( TgADF) to enhance the actin filament dynamics in the bradyzoite stage

The Toxoplasma gondii deoxyribose phosphate aldolase-like ( TgDPA) gene is expressed predominantly in bradyzoites. This finding allowed us to infer that TgDPA is important in the tachyzoite-to-bradyzoite development or maintenance of cyst structure although the function of this gene is still unknown. We conducted yeast two-hybrid screening to identify proteins interacting with TgDPA, and the actin depolymerizing factor ( TgADF) gene was obtained. Co-immunoprecipitation and a GST pull-down assay demonstrated that TgDPA interacts with TgADF. To reveal the significance of the protein–protein interaction between TgDPA and TgADF, actin polymerization and disassembly kinetics were examined. Addition of GST- TgDPA to TgADF lowered the extent of actin polymerization and enhanced the filamentous actin disassembly. These results demonstrated that this is the novel protein–protein interaction in T. gondii, and that TgDPA can enhance the activity of TgADF. This phenomenon might play an important role in T. gondii bradyzoites by affecting the actin turnover.

Glycal Formation in Crystals of Uridine Phosphorylase,

Uridine phosphorylase is a key enzyme in the pyrimidine salvage pathway. This enzyme catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate (or 2'-deoxyuridine to 2'-deoxyribose 1-phosphate). Here we report the structure of hexameric Escherichia coli uridine phosphorylase treated with 5-fluorouridine and sulfate and dimeric bovine uridine phosphorylase treated with 5-fluoro-2'-deoxyuridine or uridine, plus sulfate. In each case the electron density shows three separate species corresponding to the pyrimidine base, sulfate, and a ribosyl species, which can be modeled as a glycal. In the structures of the glycal complexes, the fluorouracil O2 atom is appropriately positioned to act as the base required for glycal formation via deprotonation at C2'. Crystals of bovine uridine phosphorylase treated with 2'-deoxyuridine and sulfate show intact nucleoside. NMR time course studies demonstrate that uridine phosphorylase can catalyze the hydrolysis of the fluorinated nucleosides in the absence of phosphate or sulfate, without the release of intermediates or enzyme inactivation. These results add a previously unencountered mechanistic motif to the body of information on glycal formation by enzymes catalyzing the cleavage of glycosyl bonds.

Potentiation of Temozolomide Cytotoxicity by Inhibition of DNA Polymerase β Is Accentuated by BRCA2 Mutation

Base excision repair (BER) plays a critical role in the repair of bases damaged by oxidative metabolism or alkylating agents, such as those commonly used in cancer therapy. Incomplete BER generates intermediates that require activation of homology-dependent DNA repair to resolve. We investigated the effects of lithocholic acid (LCA), an inhibitor of the key BER enzyme DNA polymerase beta (pol beta), in cells deficient in expression of the homology-dependent repair factor BRCA2. In vitro studies show that LCA suppresses the DNA polymerase and 5'-deoxyribose phosphate lyase activities of DNA pol beta by preventing the formation of a stable pol beta-DNA complex, reducing BER effectiveness. Cytotoxicity assays based on colony formation revealed that LCA exhibits synergism with the alkylating agent temozolomide, which engages BER through DNA methylation, and that the degree of synergism is increased in cells lacking functional BRCA2. BRCA2-deficient cells also showed heightened susceptibility to both LCA and temozolomide individually. The potentiation of temozolomide cytotoxicity by LCA owes to the conversion of single-stranded DNA breaks generated through incomplete BER of methylated nucleotides into double-stranded breaks during DNA replication, as indicated by gammaH2AX immunofluorescence. Death seems to be induced in cotreated cells through an accumulation of persistent double-stranded DNA breaks. Mutations of the BRCA2 gene have been extensively characterized and are present in various cancers, implying that inhibition of BER may offer a means to augment tumor selectivity in the use of conventional cancer therapies.

Structural Characterization of the Mammalian Deoxynucleotide N-Hydrolase Rcl and Its Stabilizing Interactions with Two Inhibitors

The gene Rcl encodes a deoxynucleoside 5'-monophosphate N-glycosidase that catalyzes the hydrolysis of the N-glycosidic bond of the nucleotide to give deoxyribose 5-phosphate and a nucleobase, preferentially a purine. This enzyme is over-expressed in several cancers, and its rate of expression is correlated to the degree of aggressiveness of tumors, which makes it a new and attractive therapeutic target. We describe here its structural characterization in the presence of two inhibitory substrate mimics. One of these ligands corresponds to the monophosphorylated form of acyclovir, which is used in the clinic. This study reveals an important ligand-induced stabilization of the dimer structure of the enzyme. The original structural features of Rcl will be helpful for designing new inhibitors.

Characterization of a Natural Mutator Variant of Human DNA Polymerase λ which Promotes Chromosomal Instability by Compromising NHEJ

BackgroundDNA polymerase lambda (Polλ) is a DNA repair polymerase, which likely plays a role in base excision repair (BER) and in non-homologous end joining (NHEJ) of DNA double-strand breaks (DSB).Principal FindingsHere, we described a novel natural allelic variant of human Polλ (hPolλ) characterized by a single nucleotide polymorphism (SNP), C/T variation in the first base of codon 438, resulting in the amino acid change Arg to Trp. In vitro enzyme activity assays of the purified W438 Polλ variant revealed that it retained both DNA polymerization and deoxyribose phosphate (dRP) lyase activities, but had reduced base substitution fidelity. Ectopic expression of the W438 hPolλ variant in mammalian cells increases mutation frequency, affects the DSB repair NHEJ pathway, and generates chromosome aberrations. All these phenotypes are dependent upon the catalytic activity of the W438 hPolλ.ConclusionsThe expression of a cancer-related natural variant of one specialized DNA polymerase can be associated to generic instability at the cromosomal level, probably due a defective NHEJ. These results establish that chromosomal aberrations can result from mutations in specialized DNA repair polymerases.

Modulation of the Structure, Catalytic Activity, and Fidelity of African Swine Fever Virus DNA Polymerase X by a Reversible Disulfide Switch

African swine fever virus polymerase X (pol X) is the smallest DNA polymerase known (174 amino acids), and its tertiary structure resembles the C-terminal half of prototypical X-family pol beta, which includes a catalytic dNTP-binding site (palm domain) and a finger domain. This structural similarity and the presence of viral genes coding for other base excision repair proteins suggest that pol X functions in a manner similar to pol beta, but inconsistencies concerning pol X catalysis have been reported. We examined the structural and functional properties of two forms of pol X using spectroscopic and kinetic analysis. Using (1)H-(15)N correlated NMR, we unambiguously demonstrated the slow interconversion of pol X between a reduced (pol X(red)) and an oxidized form (pol X(ox)), confirmed by mass spectrometry. Steady-state kinetic analysis revealed that pol X(ox), with a disulfide bond between Cys-81 and Cys-86, has approximately 10-fold lower fidelity than pol X(red) during dNTP insertion opposite a template G. The disulfide linkage is located between two beta-strands in the palm domain, near the putative dNTP-binding site. Structural alignment of pol X with a pol beta ternary structure suggests that the disulfide switch may modulate fidelity by altering the ability of the palm domain to align and stabilize the primer terminus and catalytic metal ion for deprotonation of the 3'-OH group and subsequent phosphoryl transfer. Thus, DNA polymerase fidelity is altered by the redox state of the enzyme and its related conformational changes.

DNA polymerase X from Deinococcus radiodurans implicated in bacterial tolerance to DNA damage is characterized as a short patch base excision repair polymerase

The Deinococcus radiodurans R1 genome encodes an X-family DNA repair polymerase homologous to eukaryotic DNA polymerase beta. The recombinant deinococcal polymerase X (PolX) purified from transgenic Escherichia coli showed deoxynucleotidyltransferase activity. Unlike the Klenow fragment of E. coli, this enzyme showed short patch DNA synthesis activity on heteropolymeric DNA substrate. The recombinant enzyme showed 5'-deoxyribose phosphate (5'-dRP) lyase activity and base excision repair function in vitro, with the help of externally supplied glycosylase and AP endonuclease functions. A polX disruption mutant of D. radiodurans expressing 5'-dRP lyase and a truncated polymerase domain was comparatively less sensitive to gamma-radiation than a polX deletion mutant. Both mutants showed higher sensitivity to hydrogen peroxide. Excision repair mutants of E. coli expressing this polymerase showed functional complementation of UV sensitivity. These results suggest the involvement of deinococcal polymerase X in DNA-damage tolerance of D. radiodurans, possibly by contributing to DNA double-strand break repair and base excision repair.

Cloning, expression, and characterization of a new deoxyribose 5-phosphate aldolase from Yersinia sp. EA015

A new deoC gene encoding deoxyribose 5-phosphate aldolase (DERA) was identified in Yersinia sp. EA015 isolated from soil. The DERA gene had an open reading frame (ORF) of 672 base pairs encoding 223 amino acids to yield a protein of molecular mass 24.8 kDa. The amino acid sequence was 94% identical to that of DERA from Yersinia intermedia ATCC 29909. DERA was over-expressed in Escherichia coli and purified using Ni–NTA affinity chromatography. The specific activity was 137 μmol/min/mg. The Michaelis constant ( k m value) of DERA was 9.1 mM. DERA was optimally active at pH 6.0 and 50 °C. DERA was tolerant to a high concentration (300 mM) of acetaldehyde.

Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1

Herpes simplex virus-1 is a large double-stranded DNA virus that is self-sufficient in a number of genome transactions. Hence, the virus encodes its own DNA replication apparatus and is capable of mediating recombination reactions. We recently reported that the catalytic subunit of the HSV-1 DNA polymerase (UL30) exhibits apurinic/apyrimidinic and 5'-deoxyribose phosphate lyase activities that are integral to base excision repair. Base excision repair is required to maintain genome stability as a means to counter the accumulation of unusual bases and to protect from the loss of DNA bases. Here we have reconstituted a system with purified HSV-1 and human proteins that perform all the steps of uracil DNA glycosylase-initiated base excision repair. In this system nucleotide incorporation is dependent on the HSV-1 uracil DNA glycosylase (UL2), human AP endonuclease, and the HSV-1 DNA polymerase. Completion of base excision repair can be mediated by T4 DNA ligase as well as human DNA ligase I or ligase IIIalpha-XRCC1 complex. Of these, ligase IIIalpha-XRCC1 is the most efficient. Moreover, ligase IIIalpha-XRCC1 confers specificity onto the reaction in as much as it allows ligation to occur in the presence of the HSV-1 DNA polymerase processivity factor (UL42) and prevents base excision repair from occurring with heterologous DNA polymerases. Completion of base excision repair in this system is also dependent on the incorporation of the correct nucleotide. These findings demonstrate that the HSV-1 proteins in combination with cellular factors that are not encoded by the virus are capable of performing base excision repair. These results have implications on the role of base excision repair in viral genome maintenance during lytic replication and reactivation from latency.