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1.  Elevated Levels of DNA Strand Breaks Induced by a Base Analog in the Human Cell Line with the P32T ITPA Variant 
Journal of Nucleic Acids  2010;2010:872180.
Base analogs are powerful antimetabolites and dangerous mutagens generated endogenously by oxidative stress, inflammation, and aberrant nucleotide biosynthesis. Human inosine triphosphate pyrophosphatase (ITPA) hydrolyzes triphosphates of noncanonical purine bases (i.e., ITP, dITP, XTP, dXTP, or their mimic: 6-hydroxyaminopurine (HAP) deoxynucleoside triphosphate) and thus regulates nucleotide pools and protects cells from DNA damage. We demonstrate that the model purine base analog HAP induces DNA breaks in human cells and leads to elevation of levels of ITPA. A human polymorphic allele of the ITPA, 94C->A encodes for the enzyme with a P32T amino-acid change and leads to accumulation of nonhydrolyzed ITP. The polymorphism has been associated with adverse reaction to purine base-analog drugs. The level of both spontaneous and HAP-induced DNA breaks is elevated in the cell line with the ITPA P32T variant. The results suggested that human ITPA plays a pivotal role in the protection of DNA from noncanonical purine base analogs.
PMCID: PMC2948936  PMID: 20936128
2.  Structural basis for inhibition of DNA replication by aphidicolin 
Nucleic Acids Research  2014;42(22):14013-14021.
Natural tetracyclic diterpenoid aphidicolin is a potent and specific inhibitor of B-family DNA polymerases, haltering replication and possessing a strong antimitotic activity in human cancer cell lines. Clinical trials revealed limitations of aphidicolin as an antitumor drug because of its low solubility and fast clearance from human plasma. The absence of structural information hampered the improvement of aphidicolin-like inhibitors: more than 50 modifications have been generated so far, but all have lost the inhibitory and antitumor properties. Here we report the crystal structure of the catalytic core of human DNA polymerase α (Pol α) in the ternary complex with an RNA-primed DNA template and aphidicolin. The inhibitor blocks binding of dCTP by docking at the Pol α active site and by rotating the template guanine. The structure provides a plausible mechanism for the selectivity of aphidicolin incorporation opposite template guanine and explains why previous modifications of aphidicolin failed to improve its affinity for Pol α. With new structural information, aphidicolin becomes an attractive lead compound for the design of novel derivatives with enhanced inhibitory properties for B-family DNA polymerases.
PMCID: PMC4267640  PMID: 25429975
3.  The human ITPA polymorphic variant P32T is destabilized by the unpacking of the hydrophobic core 
Journal of structural biology  2013;182(3):197-208.
Inosine triphosphate pyrophosphatase (ITPA), a key enzyme involved in maintaining the purity of cellular nucleoside triphosphate pools, specifically recognizes inosine triphosphate and xanthosine triphosphate (including the deoxyribose forms) and detoxifies them by catalyzing the hydrolysis of a phosphoanhydride bond, releasing pyrophosphate. This prevents their inappropriate use as substrates in enzymatic reactions utilizing (d)ATP or (d)GTP. A human genetic polymorphism leads to the substitution of Thr for Pro32 (P32T) and causes ITPA deficiency in erythrocytes, with heterozygotes having on average 22.5% residual activity, and homozygotes having undetectable activity. This polymorphism has been implicated in modulating patients’ response to mercaptopurines and ribavirin. Human fibroblasts containing this variant have elevated genomic instability upon treatment with base analogs. We find that the wild-type and P32T forms are dimeric in solution and in the crystal structure. This abolishes the previous speculation that the P32T change disrupts dimerization as a mechanism of inactivation. The only difference in structure from the wild-type protein is that the area surrounding Thr32 is disrupted. Phe31 is flipped from the hydrophobic core out into the solvent, leaving a hole in the hydrophobic core of the protein which likely accounts for the reduced thermal stability of P32T ITPA and ultimately leads to its susceptibility to degradation in human cells. Circular dichroism and thermal denaturation studies confirm these structural results. We propose that the dimer of P32T variant subunit with wild-type subunit is degraded in cells similarly to the P32T homodimer explaining the level of loss of ITPA activity in heterozygotes.
PMCID: PMC4212276  PMID: 23528839
Inosine triphosphate pyrophosphatase; Nucleotide pool; X-ray crystallography; Protein stability; Hydrophobic surfaces; Genomic instability
4.  The role of DNA polymerase alpha in the control of mutagenesis in Saccharomyces cerevisiae cells starved for nutrients 
Ekologicheskaia genetika  2011;9(1):53-61.
In nature, micro organisms experience numerous environmental stresses and generally grow poorly most of the time. In the last two decades it has become evident that mutations arise not only in actively dividing cells but also in non-replicating or slowly replicating cells starved for nutrients. In yeast, precise base selection and proofreading by replicative DNA polymerases δ and ε keep starvation-associated mutagenesis (SAM) at basal levels. Less is known about the role of replicative DNA polymerase α (Pol α). Here we provide evidence that Pol α is involved in the control of SAM in yeast cells starved for adenine by participation in sporadic replication and/or DNA repair under these conditions.
PMCID: PMC4197456  PMID: 25328544
Saccharomyces cerevisiae; DNA Polymerase α; starvation-associated mutagenesis
5.  ITPA (inosine triphosphate pyrophosphatase): from surveillance of nucleotide pools to human disease and pharmacogenetics 
Mutation research  2013;753(2):10.1016/j.mrrev.2013.08.001.
Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a plethora of biological reactions, from DNA and RNA synthesis to cellular signaling. An evolutionarily conserved inosine triphosphate pyrophosphatase (ITPA) removes the non-canonical purine (d)NTPs inosine triphosphate and xanthosine triphosphate by hydrolyzing them into their monophosphate form and pyrophosphate. Mutations in the ITPA orthologs in model organisms lead to genetic instability and, in mice, to severe developmental anomalies. In humans there is genetic polymorphism in ITPA. One allele leads to a proline to threonine substitution at amino acid 32 and causes varying degrees of ITPA deficiency in tissues and plays a role in patients’ response to drugs. Structural analysis of this mutant protein reveals that the protein is destabilized by the formation of a cavity in its hydrophobic core. The Pro32Thr allele is thought to cause the observed dominant negative effect because the resulting active enzyme monomer targets both homo- and heterodimers to degradation.
PMCID: PMC3827912  PMID: 23969025
nucleotide pool; ITPA gene polymorphism; pharmacogenetics; base analogs; mercaptopurines; protein stability; dominant negative
6.  Modulation of mutagenesis in eukaryotes by DNA replication fork dynamics and quality of nucleotide pools 
The rate of mutations in eukaryotes depends on a plethora of factors and is not immediately derived from the fidelity of DNA polymerases (Pols). Replication of chromosomes containing the anti-parallel strands of duplex DNA occurs through the copying of leading and lagging strand templates by a trio of Pols α, δ and ε, with the assistance of Pol ζ and Y-family Pols at difficult DNA template structures or sites of DNA damage. The parameters of the synthesis at a given location are dictated by the quality and quantity of nucleotides in the pools, replication fork architecture, transcription status, regulation of Pol switches, and structure of chromatin. The result of these transactions is a subject of survey and editing by DNA repair.
PMCID: PMC3893020  PMID: 23055184
DNA polymerases; nucleotide pools; mutagenesis; Okazaki fragments
7.  Genome-Wide Mutation Avalanches Induced in Diploid Yeast Cells by a Base Analog or an APOBEC Deaminase 
PLoS Genetics  2013;9(9):e1003736.
Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis.
Author Summary
Evolution and carcinogenesis are driven by mutations. Cells maintain constant mutation rates and can afford only transient mutagenesis bursts for adaptation. The nature of the mutational avalanches is not very clear. We sequenced the whole genomes of mutants induced in haploid and diploid yeast by nucleobase analog HAP and by DNA editing cytosine deaminase. Mutants selected in diploids are saturated with passenger mutations. Far fewer mutations are found in haploid mutants. Treatment with a mutagen without selection results in intermediate mutagenesis. The observed transient hypermutability of diploids under mutagenic insult helps to explain the wellspring of mutations that arise during evolution and carcinogenesis.
PMCID: PMC3764175  PMID: 24039593
8.  AID/APOBEC cytosine deaminase induces genome-wide kataegis 
Biology Direct  2012;7:47.
Clusters of localized hypermutation in human breast cancer genomes, named “kataegis” (from the Greek for thunderstorm), are hypothesized to result from multiple cytosine deaminations catalyzed by AID/APOBEC proteins. However, a direct link between APOBECs and kataegis is still lacking. We have sequenced the genomes of yeast mutants induced in diploids by expression of the gene for PmCDA1, a hypermutagenic deaminase from sea lamprey. Analysis of the distribution of 5,138 induced mutations revealed localized clusters very similar to those found in tumors. Our data provide evidence that unleashed cytosine deaminase activity is an evolutionary conserved, prominent source of genome-wide kataegis events.
This article was reviewed by: Professor Sandor Pongor, Professor Shamil R. Sunyaev, and Dr Vladimir Kuznetsov.
PMCID: PMC3542020  PMID: 23249472
APOBEC; Deaminase; Mutation; Kataegis; Cancer; Diploid yeast; Hypermutation
9.  Role of DNA polymerases in repeat-mediated genome instability 
Cell reports  2012;2(5):1088-1095.
Expansions of simple DNA repeats cause numerous hereditary diseases in humans. We analyzed the role of DNA polymerases in the instability of Friedreich’s ataxia (GAA)n repeats in a yeast experimental system. The elementary step of expansion corresponded to ~160 bp in the wild type strain, matching the size of Okazaki fragments in yeast. This step increased when DNA polymerase α was mutated suggesting a link between the scale of expansions and Okazaki fragment size. Expandable repeats strongly elevated the rate of mutations at substantial distances around them, a phenomenon we call repeat-induced mutagenesis (RIM). Notably, defects in the replicative DNA polymerases δ and ε strongly increased rates for both repeat expansions and RIM. The increases in repeat-mediated instability observed in DNA polymerase δ mutants depended on translesion DNA polymerases. We conclude that repeat expansions and RIM are two sides of the same replicative mechanism.
PMCID: PMC3513503  PMID: 23142667
10.  Crystal structure of the C-terminal domain of human DNA primase large subunit 
Cell Cycle  2011;10(6):926-931.
DNA polymerases cannot synthesize DNA without a primer, and DNA primase is the only specialized enzyme capable of de novo synthesis of short RNA primers. In eukaryotes, primase functions within a heterotetrameric complex in concert with a tightly bound DNA polymerase α (Pol α). In humans, the Pol α part is comprised of a catalytic subunit (p180) and an accessory subunit B (p70), and the primase part consists of a small catalytic subunit (p49) and a large essential subunit (p58). The latter subunit participates in primer synthesis, counts the number of nucleotides in a primer, assists the release of the primer-template from primase and transfers it to the Pol α active site. Recently reported crystal structures of the C-terminal domains of the yeast and human enzymes' large subunits provided critical information related to their structure, possible sites for binding of nucleotides and template DNA, as well as the overall organization of eukaryotic primases. However, the structures also revealed a difference in the folding of their proposed DNA-binding fragments, raising the possibility that yeast and human proteins are functionally different. Here we report new structure of the C-terminal domain of the human primase p58 subunit. This structure exhibits a fold similar to a fold reported for the yeast protein but different than a fold reported for the human protein. Based on a comparative analysis of all three C-terminal domain structures, we propose a mechanism of RNA primer length counting and dissociation of the primer-template from primase by a switch in conformation of the ssDNA-binding region of p58.
PMCID: PMC3100874  PMID: 21346410
DNA primase; prim1; prim2; replication; 4Fe-4S cluster; crystal structure; DNA polymerase α
11.  Pivotal Role of Inosine Triphosphate Pyrophosphatase in Maintaining Genome Stability and the Prevention of Apoptosis in Human Cells 
PLoS ONE  2012;7(2):e32313.
Pure nucleotide precursor pools are a prerequisite for high-fidelity DNA replication and the suppression of mutagenesis and carcinogenesis. ITPases are nucleoside triphosphate pyrophosphatases that clean the precursor pools of the non-canonical triphosphates of inosine and xanthine. The precise role of the human ITPase, encoded by the ITPA gene, is not clearly defined. ITPA is clinically important because a widespread polymorphism, 94C>A, leads to null ITPase activity in erythrocytes and is associated with an adverse reaction to thiopurine drugs. We studied the cellular function of ITPA in HeLa cells using the purine analog 6-N hydroxylaminopurine (HAP), whose triphosphate is also a substrate for ITPA. In this study, we demonstrate that ITPA knockdown sensitizes HeLa cells to HAP-induced DNA breaks and apoptosis. The HAP-induced DNA damage and cytotoxicity observed in ITPA knockdown cells are rescued by an overexpression of the yeast ITPase encoded by the HAM1 gene. We further show that ITPA knockdown results in elevated mutagenesis in response to HAP treatment. Our studies reveal the significance of ITPA in preventing base analog-induced apoptosis, DNA damage and mutagenesis in human cells. This implies that individuals with defective ITPase are predisposed to genome damage by impurities in nucleotide pools, which is drastically augmented by therapy with purine analogs. They are also at an elevated risk for degenerative diseases and cancer.
PMCID: PMC3288088  PMID: 22384212
12.  Replication Protein A (RPA) Hampers the Processive Action of APOBEC3G Cytosine Deaminase on Single-Stranded DNA 
PLoS ONE  2011;6(9):e24848.
Editing deaminases have a pivotal role in cellular physiology. A notable member of this superfamily, APOBEC3G (A3G), restricts retroviruses, and Activation Induced Deaminase (AID) generates antibody diversity by localized deamination of cytosines in DNA. Unconstrained deaminase activity can cause genome-wide mutagenesis and cancer. The mechanisms that protect the genomic DNA from the undesired action of deaminases are unknown. Using the in vitro deamination assays and expression of A3G in yeast, we show that replication protein A (RPA), the eukaryotic single-stranded DNA (ssDNA) binding protein, severely inhibits the deamination activity and processivity of A3G.
Principal Findings/Methodology
We found that mutations induced by A3G in the yeast genomic reporter are changes of a single nucleotide. This is unexpected because of the known property of A3G to catalyze multiple deaminations upon one substrate encounter event in vitro. The addition of recombinant RPA to the oligonucleotide deamination assay severely inhibited A3G activity. Additionally, we reveal the inverse correlation between RPA concentration and the number of deaminations induced by A3G in vitro on long ssDNA regions. This resembles the “hit and run” single base substitution events observed in yeast.
Our data suggest that RPA is a plausible antimutator factor limiting the activity and processivity of editing deaminases in the model yeast system. Because of the similar antagonism of yeast RPA and human RPA with A3G in vitro, we propose that RPA plays a role in the protection of the human genome cell from A3G and other deaminases when they are inadvertently diverged from their natural targets. We propose a model where RPA serves as one of the guardians of the genome that protects ssDNA from the destructive processive activity of deaminases by non-specific steric hindrance.
PMCID: PMC3174200  PMID: 21935481
13.  DNA Polymerases at the Eukaryotic Fork - 20 Years Later 
Mutation research  2009;685(1-2):45.
Function of the eukaryotic genome depends on efficient and accurate replication of anti-parallel DNA strands. Eukaryotic DNA polymerases have different properties adapted to perform a wide spectrum of DNA transactions. Here we focus on major players in the bulk replication, DNA polymerases of the B-family. We review the organization of the replication fork in eukaryotes in a historical perspective, analyze contemporary models and propose a new integrative model of the fork.
PMCID: PMC2822129  PMID: 19682465
replication fork; DNA polymerases; replication origins
14.  Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota 
PLoS ONE  2011;6(1):e16612.
Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.
PMCID: PMC3031609  PMID: 21304950
15.  Mismatch Repair–Independent Increase in Spontaneous Mutagenesis in Yeast Lacking Non-Essential Subunits of DNA Polymerase ε 
PLoS Genetics  2010;6(11):e1001209.
Yeast DNA polymerase ε (Pol ε) is a highly accurate and processive enzyme that participates in nuclear DNA replication of the leading strand template. In addition to a large subunit (Pol2) harboring the polymerase and proofreading exonuclease active sites, Pol ε also has one essential subunit (Dpb2) and two smaller, non-essential subunits (Dpb3 and Dpb4) whose functions are not fully understood. To probe the functions of Dpb3 and Dpb4, here we investigate the consequences of their absence on the biochemical properties of Pol ε in vitro and on genome stability in vivo. The fidelity of DNA synthesis in vitro by purified Pol2/Dpb2, i.e. lacking Dpb3 and Dpb4, is comparable to the four-subunit Pol ε holoenzyme. Nonetheless, deletion of DPB3 and DPB4 elevates spontaneous frameshift and base substitution rates in vivo, to the same extent as the loss of Pol ε proofreading activity in a pol2-4 strain. In contrast to pol2-4, however, the dpb3Δdpb4Δ does not lead to a synergistic increase of mutation rates with defects in DNA mismatch repair. The increased mutation rate in dpb3Δdpb4Δ strains is partly dependent on REV3, as well as the proofreading capacity of Pol δ. Finally, biochemical studies demonstrate that the absence of Dpb3 and Dpb4 destabilizes the interaction between Pol ε and the template DNA during processive DNA synthesis and during processive 3′ to 5′exonucleolytic degradation of DNA. Collectively, these data suggest a model wherein Dpb3 and Dpb4 do not directly influence replication fidelity per se, but rather contribute to normal replication fork progression. In their absence, a defective replisome may more frequently leave gaps on the leading strand that are eventually filled by Pol ζ or Pol δ, in a post-replication process that generates errors not corrected by the DNA mismatch repair system.
Author Summary
The high fidelity of DNA replication is safeguarded by the accuracy of nucleotide selection by DNA polymerases, proofreading activity of the replicative polymerases, and the DNA mismatch repair system. Errors made by replicative polymerases are corrected by mismatch repair, and inactivation of the mismatch repair system results in a multiplicative increase in error rates when combined with a proofreading deficient allele of a replicative polymerase. In this study, we demonstrate that the deletion of two non-essential genes encoding for two subunits of Pol ε give an increased mutation rate due to increased synthesis by the error-prone DNA polymerase ζ. Surprisingly, there was no multiplicative increase in error rates when the mismatch repair system was inactivated. We propose that the deletion of DPB3 and DPB4 gives a defective replisome, which in turn gives increased synthesis, in part, by Pol ζ during an error-prone post-replication process that is not efficiently repaired by the mismatch repair system.
PMCID: PMC2987839  PMID: 21124948
16.  Functional Study of the P32T ITPA Variant Associated with Drug Sensitivity in Humans 
Journal of molecular biology  2009;392(3):602-613.
Sanitization of the cellular nucleotide pools from mutagenic base analogs is necessary for the accuracy of transcription and replication of genetic material and plays a substantial role in cancer prevention. The undesirable mutagenic, recombinogenic and toxic incorporation of purine base analogs (i.e. ITP, dITP, XTP, dXTP or 6-hydroxyaminopurine (HAP) deoxynucleoside triphosphate) into nucleic acids is prevented by inosine triphosphate pyrophosphatase (ITPA). The ITPA gene is a highly conserved, moderately expressed gene. Defects in ITPA orthologs in model organisms cause severe sensitivity to HAP and chromosome fragmentation. A human polymorphic allele 94C->A encodes for the enzyme with a P32T amino acid change and leads to accumulation of non-hydrolyzed ITP. ITPase activity is not detected in erythrocytes of these patients. The P32T polymorphism has also been associated with adverse sensitivity to purine base analog drugs. We have found that the ITPA-P32T mutant is a dimer in solution, as is wild-type ITPA, and has normal ITPA activity in vitro, but the melting point of ITPA-P32T is 5 degrees C lower than that of wild-type. ITPA-P32T is also fully functional in vivo in model organisms as determined by a HAP mutagenesis assay and its complementation of a bacterial ITPA defect. The amount of ITPA protein detected by western blot is severely diminished in a human fibroblast cell line with the 94C->A change. We propose that the P32T mutation exerts its effect in certain human tissues by cumulative effects of destabilization of transcripts, protein stability and availability.
PMCID: PMC2745931  PMID: 19631656
17.  Crystallization and preliminary crystallographic analysis of the complex of the second and third regulatory subunits of human Pol δ 
The cloning, expression, purification and crystallization of the complex of the second and third regulatory subunits of human Pol δ are reported. The crystals were characterized and an X-ray diffraction data set was collected to a resolution of 3 Å.
Human DNA polymerase δ (Pol δ) consists of four subunits: p125, p50, p66 and p12. A heterodimer containing a His-tagged p50 subunit (p50) and a p50-interacting domain of the p66 subunit (p66N) was crystallized. The crystal was in the form of a prism with a rhombic cross-section and belonged to space group P21. The crystal had unit-cell parameters a = 95.13, b = 248.54, c = 103.46 Å, β = 106.94° and diffracted to a resolution of 3 Å. Four molecules of p50–p66N in an asymmetric unit corresponded to a crystal solvent content of 72.2%.
PMCID: PMC2531283  PMID: 18765914
human DNA polymerase δ; Pol δ; p50 subunit; p66 subunit
18.  Evidence for Extrinsic Exonucleolytic Proofreading 
Cell cycle (Georgetown, Tex.)  2006;5(9):958-962.
Exonucleolytic proofreading of DNA synthesis errors is one of the major determinants of genome stability. However, many DNA transactions that contribute to genome stability require synthesis by polymerases that naturally lack intrinsic 3' exonuclease activity and some of which are highly inaccurate. Here we discuss evidence that errors made by these polymerases may be edited by a separate 3' exonuclease, and we consider how such extrinsic proofreading may differ from proofreading by exonucleases that are intrinsic to replicative DNA polymerases.
PMCID: PMC2907748  PMID: 16687920
proofreading; DNA replication fidelity; DNA repair; mutagenesis; base substitutions; DNA polymerase; exonuclease
19.  YcbX and yiiM, two novel determinants for resistance of E. coli to N-hydroxylated base analogs 
Molecular microbiology  2008;68(1):51-65.
We have shown previously that lack of molybdenum cofactor (MoCo) in Escherichia coli leads to hypersensitivity to the mutagenic and toxic effects of N-hydroxylated base analogs, such as 6-N-hydroxylaminopurine (HAP). However, the nature of the MoCo-dependent mechanism is unknown, as inactivation of all known and putative E. coli molybdoenzymes does not produce any sensitivity. Presently, we report on the isolation and characterization of two novel HAP-hypersensitive mutants carrying defects in the ycbX or yiiM open reading frames. Genetic analysis suggests that the two genes operate within the MoCo-dependent pathway. In the absence of the ycbX- and yiiM-dependent pathways, biotin sulfoxide reductase (BisC) plays also a role in the detoxification pathway. YcbX and YiiM are hypothetical members of the MOSC protein superfamily, which contain the C-terminal domain (MOSC) of the eukaryotic MoCo sulfurases. However, deletion of ycbX or yiiM did not affect the activity of human xanthine dehydrogenase expressed in E. coli, suggesting that the role of YcbX and YiiM proteins is not related to MoCo sulfuration. Instead, YcbX and YiiM may represent novel MoCo-dependent enzymatic activities. We also demonstrate that the MoCo/YcbX/YiiM-dependent detoxification of HAP proceeds by reduction to adenine.
PMCID: PMC2740630  PMID: 18312271
20.  Evolution of DNA polymerases: an inactivated polymerase-exonuclease module in Pol ε and a chimeric origin of eukaryotic polymerases from two classes of archaeal ancestors 
Biology Direct  2009;4:11.
Evolution of DNA polymerases, the key enzymes of DNA replication and repair, is central to any reconstruction of the history of cellular life. However, the details of the evolutionary relationships between DNA polymerases of archaea and eukaryotes remain unresolved.
We performed a comparative analysis of archaeal, eukaryotic, and bacterial B-family DNA polymerases, which are the main replicative polymerases in archaea and eukaryotes, combined with an analysis of domain architectures. Surprisingly, we found that eukaryotic Polymerase ε consists of two tandem exonuclease-polymerase modules, the active N-terminal module and a C-terminal module in which both enzymatic domains are inactivated. The two modules are only distantly related to each other, an observation that suggests the possibility that Pol ε evolved as a result of insertion and subsequent inactivation of a distinct polymerase, possibly, of bacterial descent, upstream of the C-terminal Zn-fingers, rather than by tandem duplication. The presence of an inactivated exonuclease-polymerase module in Pol ε parallels a similar inactivation of both enzymatic domains in a distinct family of archaeal B-family polymerases. The results of phylogenetic analysis indicate that eukaryotic B-family polymerases, most likely, originate from two distantly related archaeal B-family polymerases, one form giving rise to Pol ε, and the other one to the common ancestor of Pol α, Pol δ, and Pol ζ. The C-terminal Zn-fingers that are present in all eukaryotic B-family polymerases, unexpectedly, are homologous to the Zn-finger of archaeal D-family DNA polymerases that are otherwise unrelated to the B family. The Zn-finger of Polε shows a markedly greater similarity to the counterpart in archaeal PolD than the Zn-fingers of other eukaryotic B-family polymerases.
Evolution of eukaryotic DNA polymerases seems to have involved previously unnoticed complex events. We hypothesize that the archaeal ancestor of eukaryotes encoded three DNA polymerases, namely, two distinct B-family polymerases and a D-family polymerase all of which contributed to the evolution of the eukaryotic replication machinery. The Zn-finger might have been acquired from PolD by the B-family form that gave rise to Pol ε prior to or in the course of eukaryogenesis, and subsequently, was captured by the ancestor of the other B-family eukaryotic polymerases. The inactivated polymerase-exonuclease module of Pol ε might have evolved by fusion with a distinct polymerase, rather than by duplication of the active module of Pol ε, and is likely to play an important role in the assembly of eukaryotic replication and repair complexes.
This article was reviewed by Patrick Forterre, Arcady Mushegian, and Chris Ponting. For the full reviews, please go to the Reviewers' Reports section.
PMCID: PMC2669801  PMID: 19296856
21.  X-ray structure of the complex of regulatory subunits of human DNA polymerase δ 
Cell cycle (Georgetown, Tex.)  2008;7(19):3026-3036.
The eukaryotic DNA polymerase δ (Pol δ) participates in genome replication, homologous recombination, DNA repair and damage tolerance. Regulation of the plethora of Pol δ functions depends on the interaction between the second (p50) and third (p66) non-catalytic subunits. We report the crystal structure of p50•p66N complex featuring oligonucleotide binding and phosphodiesterase domains in p50 and winged helix-turn-helix N-terminal domain in p66. Disruption of the interaction between the yeast orthologs of p50 and p66 by strategic amino acid changes leads to cold-sensitivity, sensitivity to hydroxyurea and to reduced UV mutagenesis, mimicking the phenotypes of strains where the third subunit of Pol δ is absent. The second subunits of all B family replicative DNA polymerases in archaea and eukaryotes, except Pol δ, share a three-domain structure similar to p50•p66N, raising the possibility that a portion of the gene encoding p66 was derived from the second subunit gene relatively late in evolution.
PMCID: PMC2605013  PMID: 18818516
DNA polymerase δ; Pol δ; p50; p66; Pol31; Pol32; OB; Myb; phosphodiesterase; human; yeast
22.  Mutator alleles of yeast DNA polymerase ζ 
DNA repair  2007;6(12):1829-1838.
The yeast REV3 gene encodes the catalytic subunit of DNA polymerase zeta (pol ζ), a B family polymerase that performs mutagenic DNA synthesis in cells. To probe pol ζ mutagenic functions, we generated six mutator alleles of REV3 with amino acid replacements for Leu979, a highly conserved residue inferred to be at the pol ζ active site. Replacing Leu979 with Gly, Val, Asn, Lys, Met or Phe resulted in yeast strains with elevated UV-induced mutant frequencies. While four of these strains had reduced survival following UV irradiation, the rev3-L979F and rev3-L979M strains had normal survival, suggesting retention of pol ζ catalytic activity. UV mutagenesis in the rev3-L979F background was increased when photoproduct bypass by pol η was eliminated by deletion of RAD30. The rev3-L979F mutation had little to no effect on mutagenesis in an ogg1Δ background, which cannot repair 8-oxo-guanine in DNA. UV-induced can1 mutants from rev3-L979F and rad30Δrev3-L979F strains primarily contained base substitutions and complex mutations, suggesting error-prone bypass of UV photoproducts by L979F pol ζ. Spontaneous mutation rates in rev3-L979F and rev3-L979M strains are elevated by about 2-fold overall and by 2- to 8-fold for C to G transversions and complex mutations, both of which are known to be generated by wild-type pol ζ in vitro. These results indicate that Rev3p-Leu979 replacements reduce the fidelity of DNA synthesis by yeast pol ζ in vivo. In conjunction with earlier studies, the data establish that the conserved amino acid at the active site location occupied by Leu979 is critical for the fidelity of all four yeast B family polymerases. Reduced fidelity with retention of robust polymerase activity suggests that the homologous rev3-L979F allele may be useful for analyzing pol ζ functions in mammals, where REV3 deletion is lethal.
PMCID: PMC2128049  PMID: 17715002
pol ζ; translesion synthesis; mutagenesis; yeast
23.  Structure of the orthorhombic form of human inosine triphosphate pyrophosphatase 
X-ray crystallographic analysis of human inosine triphosphate pyrophosphohydrolase provided the secondary structure and active-site structure at 1.6 Å resolution in an orthorhombic crystal form. The structure gives a framework for future structure–function studies employing site-directed mutagenesis and for the identification of substrate/product-binding sites.
The structure of human inosine triphosphate pyrophosphohydrolase (ITPA) has been determined using diffraction data to 1.6 Å resolution. ITPA contributes to the accurate replication of DNA by cleansing cellular dNTP pools of mutagenic nucleotide purine analogs such as dITP or dXTP. A similar high-resolution unpublished structure has been deposited in the Protein Data Bank from a monoclinic and pseudo-merohedrally twinned crystal. Here, cocrystallization of ITPA with a molar ratio of XTP appears to have improved the crystals by eliminating twinning and resulted in an orthorhombic space group. However, there was no evidence for bound XTP in the structure. Comparison with substrate-bound NTPase from a thermophilic organism predicts the movement of residues within helix α1, the loop before α6 and helix α7 to cap off the active site when substrate is bound.
PMCID: PMC2225220  PMID: 17077483
inosine triphosphate pyrophosphohydrolase
24.  A highly conserved family of inactivated archaeal B family DNA polymerases 
Biology Direct  2008;3:32.
A widespread and highly conserved family of apparently inactivated derivatives of archaeal B-family DNA polymerases is described. Phylogenetic analysis shows that the inactivated forms comprise a distinct clade among archaeal B-family polymerases and that, within this clade, Euryarchaea and Crenarchaea are clearly separated from each other and from a small group of bacterial homologs. These findings are compatible with an ancient duplication of the DNA polymerase gene followed by inactivation and parallel loss in some of the lineages although contribution of horizontal gene transfer cannot be ruled out. The inactivated derivative of the archaeal DNA polymerase could form a complex with the active paralog and play a structural role in DNA replication.
This article was reviewed by Purificacion Lopez-Garcia and Chris Ponting. For the full reviews, please go to the Reviewers' Reports section.
PMCID: PMC2527604  PMID: 18684330
25.  Correction: Evidence for interplay among yeast replicative DNA polymerases alpha, delta and epsilon from studies of exonuclease and polymerase active site mutations 
BMC Biology  2007;5:27.
This article has been published as a correction for an error in the manuscript of Pavlov et al BMC Biology 2004, 2:11.
PMCID: PMC1913497

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