Biomedical subjects

Youri I Pavlov

Publications and source records attributed to Youri I Pavlov.

At least 19 recordsLinked to original sources

PubMed2006

Structure of the orthorhombic form of human inosine triphosphate pyrophosphatase.

The structure of human inosine triphosphate pyrophosphohydrolase (ITPA) has been determined using diffraction data to 1.6 A 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 A similar high-resolution unpublished structure has been deposited in the Protein Data Bank from a monoclinic and pseudo-merohedrally twinned crystal.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 alpha1, the loop before alpha6 and helix alpha7 to cap off the active site when substrate is bound.

Crystallography, X-Ray↗

PubMed2006

Evidence for extrinsic exonucleolytic proofreading.

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.

DNA↗

PubMed2006

Roles of DNA polymerases in replication, repair, and recombination in eukaryotes.

The functioning of the eukaryotic genome depends on efficient and accurate DNA replication and repair. The process of replication is complicated by the ongoing decomposition of DNA and damage of the genome by endogenous and exogenous factors. DNA damage can alter base coding potential resulting in mutations, or block DNA replication, which can lead to double-strand breaks (DSB) and to subsequent chromosome loss. Replication is coordinated with DNA repair systems that operate in cells to remove or tolerate DNA lesions. DNA polymerases can serve as sensors in the cell cycle checkpoint pathways that delay cell division until damaged DNA is repaired and replication is completed. Eukaryotic DNA template-dependent DNA polymerases have different properties adapted to perform an amazingly wide spectrum of DNA transactions. In this review, we discuss the structure, the mechanism, and the evolutionary relationships of DNA polymerases and their possible functions in the replication of intact and damaged chromosomes, DNA damage repair, and recombination.

Amino Acid Sequence↗

PubMed2005

The cytidine deaminase AID exhibits similar functional properties in yeast and mammals.

A recent work published in Molecular Immunology examined the editing activity of activation-induced deaminase (AID) in yeast (Krause, K., Marcu, K.B., Greeve, J., 2006. The cytidine deaminases AID and APOBEC-1 exhibit distinct functional properties in a novel yeast selectable system. Mol. Immunol.). It was proposed that expression of AID in yeast is not sufficient for the generation of point mutations in a highly transcribed gene due to the lack of cofactors for AID-induced somatic hypermutation, which are unique to B cells. It was suggested that, on its own, AID does not have an intrinsic specificity for its target sequences. However, it has been shown previously that expression of the human AID gene in yeast was moderately mutagenic in a wild-type strain and highly mutagenic in an ung1 uracil-DNA glycosylase-deficient strain (Mayorov, V.I., Rogozin, I.B., Adkison, L.R., Frahm, C.R., Kunkel T.A., Pavlov Y. I., 2005. Expression of human AID in yeast induces mutations in context similar to the context of somatic hypermutation at G-C pairs in immunoglobulin genes. BMC Immunol. 6, 10; Poltoratsky, V.P., Wilson, S.H., Kunkel, T.A., Pavlov, Y.I., 2004. Recombinogenic phenotype of human activation-induced cytosine deaminase. J. Immunol. 172, 4308-4313). The vast majority of mutations were at G-C pairs. Mutations showed a clear DNA sequence context specificity which resembled the specificity of somatic hypermutation at G-C pairs in immunoglobulin genes and AID mutation specificity in vitro. The inability to detect mutator effects of AID by Krause et al. is likely to be caused by the use of the wild-type yeast strain and a small sample of clones examined for the presence of mutations. In addition, we show that non-uniformity of the mutation hotspot distribution is a factor potentially decreasing the chances of detecting mutations.

Animals↗

PubMed2005

APOBEC4, a new member of the AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases predicted by computational analysis.

Using iterative database searches, we identified a new subfamily of the AID/APOBEC family of RNA/DNA editing cytidine deaminases. The new subfamily, which is represented by readily identifiable orthologs in mammals, chicken, and frog, but not fishes, was designated APOBEC4. The zinc-coordinating motifs involved in catalysis and the secondary structure of the APOBEC4 deaminase domain are evolutionarily conserved, suggesting that APOBEC4 proteins are active polynucleotide (deoxy)cytidine deaminases. In reconstructed maximum likelihood phylogenetic trees, APOBEC4 forms a distinct clade with a high statistical support. APOBEC4 and APOBEC1 are joined in a moderately supported cluster clearly separated from AID, APOBEC2 and APOBEC3 subfamilies. In mammals, APOBEC4 is expressed primarily in testis which suggests the possibility that it is an editing enzyme for mRNAs involved in spermatogenesis.

APOBEC Deaminases↗

PubMed2005

Saccharomyces cerevisiae DNA polymerase delta: high fidelity for base substitutions but lower fidelity for single- and multi-base deletions.

Eukaryotic DNA polymerase delta (Pol delta) plays an essential role in replicating large nuclear genomes, a process that must be accurate to maintain stability over many generations. Based on kinetic studies of insertion of individual dNTPs opposite a template guanine, Pol delta is believed to have high selectivity for inserting correct nucleotides. This high selectivity, in conjunction with an intrinsic 3'-exonuclease activity, implies that Pol delta should have high base substitution fidelity. Here we demonstrate that the wild type Saccharomyces cerevisiae three-subunit Pol delta does indeed have high base substitution fidelity for the 12 possible base-base mismatches, producing on average less than 1.3 stable misincorporations/100,000 nucleotides polymerized. Measurements with exonuclease-deficient Pol delta confirm the high nucleotide selectivity of the polymerase and further indicate that proofreading enhances the base substitution fidelity of the wild type enzyme by at least 60-fold. However, Pol delta inefficiently proofreads single nucleotide deletion mismatches in homopolymeric runs, such that the error rate is 30 single nucleotide deletions/100,000 nucleotides polymerized. Moreover, wild type Pol delta frequently deletes larger numbers of nucleotides between distantly spaced direct repeats of three or more base pairs. Although wild type Pol delta and Pol epsilon both have high base substitution fidelity, Pol delta is much less accurate than Pol epsilon for deletions involving repetitive sequences. Thus, strand slippage during replication by wild type Pol delta may be a primary source of insertion and deletion mutagenesis in eukaryotic genomes.

Base Sequence↗

PubMed2005

Expression of human AID in yeast induces mutations in context similar to the context of somatic hypermutation at G-C pairs in immunoglobulin genes.

BACKGROUND: Antibody genes are diversified by somatic hypermutation (SHM), gene conversion and class-switch recombination. All three processes are initiated by the activation-induced deaminase (AID). According to a DNA deamination model of SHM, AID converts cytosine to uracil in DNA sequences. The initial deamination of cytosine leads to mutation and recombination in pathways involving replication, DNA mismatch repair and possibly base excision repair. The DNA sequence context of mutation hotspots at G-C pairs during SHM is DGYW/WRCH (G-C is a hotspot position, R = A/G, Y = T/C, W = A/T, D = A/G/T). RESULTS: To investigate the mechanisms of AID-induced mutagenesis in a model system, we studied the genetic consequences of AID expression in yeast. We constructed a yeast vector with an artificially synthesized human AID gene insert using codons common to highly expressed yeast genes. We found that expression of the artificial hAIDSc gene was moderately mutagenic in a wild-type strain and highly mutagenic in an ung1 uracil-DNA glycosylase-deficient strain. A majority of mutations were at G-C pairs. In the ung1 strain, C-G to T-A transitions were found almost exclusively, while a mixture of transitions with 12% transversions was characteristic in the wild-type strain. In the ung1 strain mutations that could have originated from deamination of the transcribed stand were found more frequently. In the wild-type strain, the strand bias was reversed. DGYW/WRCH motifs were preferential sites of mutations. CONCLUSION: The results are consistent with the hypothesis that AID-mediated deamination of DNA is a major cause of mutations at G-C base pairs in immunoglobulin genes during SHM. The sequence contexts of mutations in yeast induced by AID and those of somatic mutations at G-C pairs in immunoglobulin genes are significantly similar. This indicates that the intrinsic substrate specificity of AID itself is a primary determinant of mutational hotspots at G-C base pairs during SHM.

Amino Acid Transport Systems, Basic↗

PubMed2005

Genome-wide screening for genes whose deletions confer sensitivity to mutagenic purine base analogs in yeast.

BACKGROUND: N-hydroxylated base analogs, such as 6-hydroxylaminopurine (HAP) and 2-amino-6-hydroxylaminopurine (AHA), are strong mutagens in various organisms due to their ambiguous base-pairing properties. The systems protecting cells from HAP and related noncanonical purines in Escherichia coli include specialized deoxyribonucleoside triphosphatase RdgB, DNA repair endonuclease V, and a molybdenum cofactor-dependent system. Fewer HAP-detoxification systems have been identified in yeast Saccharomyces cerevisiae and other eukaryotes. Cellular systems protecting from AHA are unknown. In the present study, we performed a genome-wide search for genes whose deletions confer sensitivity to HAP and AHA in yeast. RESULTS: We screened the library of yeast deletion mutants for sensitivity to the toxic and mutagenic action of HAP and AHA. We identified novel genes involved in the genetic control of base analogs sensitivity, including genes controlling purine metabolism, cytoskeleton organization, and amino acid metabolism. CONCLUSION: We developed a method for screening the yeast deletion library for sensitivity to the mutagenic and toxic action of base analogs and identified 16 novel genes controlling pathways of protection from HAP. Three of them also protect from AHA.

Adaptation, Biological↗

PubMed2005

From context-dependence of mutations to molecular mechanisms of mutagenesis.

Base Sequence↗

PubMed2004

The exceptionally high rate of spontaneous mutations in the polymerase delta proofreading exonuclease-deficient Saccharomyces cerevisiae strain starved for adenine.

BACKGROUND: Mutagenesis induced in the yeast Saccharomyces cerevisiae by starvation for nutrilites is a well-documented phenomenon of an unknown mechanism. We have previously shown that the polymerase delta proofreading activity controls spontaneous mutagenesis in cells starved for histidine. To obtain further information, we compared the effect of adenine starvation on mutagenesis in wild-type cells and, in cells lacking the proofreading activity of polymerase delta (phenotype Exo-, mutation pol3-01). RESULTS: Ade+ revertants accumulated at a very high rate on adenine-free plates so that their frequency on day 16 after plating was 1.5 x 10(-4) for wild-type and 1.0 x 10(-2) for the Exo- strain. In the Exo- strain, all revertants arising under adenine starvation are suppressors of the original mutation, most possessed additional nutritional requirements, and 50% of them were temperature sensitive. CONCLUSIONS: Adenine starvation is highly mutagenic in yeast. The deficiency in the polymerase delta proofreading activity in strains with the pol3-01 mutation leads to a further 66-fold increase of the rate of mutations. Our data suggest that adenine starvation induces genome-wide hyper-mutagenesis in the Exo- strain.

Adenine↗

PubMed2004

Evidence for interplay among yeast replicative DNA polymerases alpha, delta and epsilon from studies of exonuclease and polymerase active site mutations.

BACKGROUND: DNA polymerase epsilon (Pol epsilon) is essential for S-phase replication, DNA damage repair and checkpoint control in yeast. A pol2-Y831A mutation leading to a tyrosine to alanine change in the Pol epsilon active site does not cause growth defects and confers a mutator phenotype that is normally subtle but strong in a mismatch repair-deficient strain. Here we investigate the mechanism responsible for the mutator effect. RESULTS: Purified four-subunit Y831A Pol epsilon turns over more deoxynucleoside triphosphates to deoxynucleoside monophosphates than does wild-type Pol epsilon, suggesting altered coordination between the polymerase and exonuclease active sites. The pol2-Y831A mutation suppresses the mutator effect of the pol2-4 mutation in the exonuclease active site that abolishes proofreading by Pol epsilon, as measured in haploid strain with the pol2-Y831A,4 double mutation. Analysis of mutation rates in diploid strains reveals that the pol2-Y831A allele is recessive to pol2-4. In addition, the mutation rates of strains with the pol2-4 mutation in combination with active site mutator mutations in Pol delta and Pol alpha suggest that Pol epsilon may proofread certain errors made by Pol alpha and Pol delta during replication in vivo. CONCLUSIONS: Our data suggest that Y831A replacement in Pol epsilon reduces replication fidelity and its participation in chromosomal replication, but without eliminating an additional function that is essential for viability. This suggests that other polymerases can substitute for certain functions of polymerase epsilon.

Binding Sites↗

PubMed2004

Recombinogenic phenotype of human activation-induced cytosine deaminase.

Class switch recombination, gene conversion, and somatic hypermutation that diversify rearranged Ig genes to produce various classes of high affinity Abs are dependent on the enzyme activation-induced cytosine deaminase (AID). Evidence suggests that somatic hypermutation is due to error-prone DNA repair that is initiated by AID-mediated deamination of cytosine in DNA, whereas the mechanism by which AID controls recombination remains to be elucidated. In this study, using a yeast model system, we have observed AID-dependent recombination. Expression of human AID in wild-type yeast is mutagenic for G-C to A-T transitions, and as expected, this mutagenesis is increased upon inactivation of uracil-DNA glycosylase. AID expression also strongly induces intragenic mitotic recombination, but only in a strain possessing uracil-DNA glycosylase. Thus, the initial step of base excision repair is required for AID-dependent recombination and is a branch point for either hypermutagenesis or recombination.

Base Pair Mismatch↗

PubMed2003

Roles of Saccharomyces cerevisiae DNA polymerases Poleta and Polzeta in response to irradiation by simulated sunlight.

Sunlight causes lesions in DNA that if unrepaired and inaccurately replicated by DNA polymerases yield mutations that result in skin cancer in humans. Two enzymes involved in translesion synthesis (TLS) of UV-induced photolesions are DNA polymerase eta (Poleta) and polymerase zeta (Polzeta), encoded by the RAD30A and REV3 genes, respectively. Previous studies have investigated the TLS roles of these polymerases in human and yeast cells irradiated with monochromatic, short wavelength UVC radiation (254 nm). However, less is known about cellular responses to solar radiation, which is of higher and mixed wavelengths (310-1100 nm) and produces a different spectrum of DNA lesions, including Dewar photoproducts and oxidative lesions. Here we report on the comparative cytotoxic and mutagenic effects of simulated sunlight (SSL) and UVC radiation on yeast wild-type, rad30Delta, rev3Delta and rev3Delta rad30Delta strains. The results with SSL support several previous interpretations on the roles of these two polymerases in TLS of photodimers and (6-4) photoproducts derived from studies with UVC. They further suggest that Poleta participates in the non-mutagenic bypass of SSL-dependent cytosine-containing Dewar photoproducts and 8-oxoguanine, while Polzeta is mainly responsible for the mutagenic bypass of all types of Dewar photoproducts. They also suggest that in the absence of Polzeta, Poleta contributes to UVC- and SSL-induced mutagenesis, possibly by the bypass of photodimers containing deaminated cytosine.

Base Sequence↗

PubMed2003

Unique error signature of the four-subunit yeast DNA polymerase epsilon.

We have purified wild type and exonuclease-deficient four-subunit DNA polymerase epsilon (Pol epsilon) complex from Saccharomyces cerevisiae and analyzed the fidelity of DNA synthesis by the two enzymes. Wild type Pol epsilon synthesizes DNA accurately, generating single-base substitutions and deletions at average error rates of </=2 x 10-5 and </=5 x 10-7, respectively. Pol epsilon lacking 3' --> 5' exonuclease activity is less accurate to a degree suggesting that wild type Pol epsilon proofreads at least 92% of base substitution errors and at least 99% of frameshift errors made by the polymerase. Surprisingly the base substitution fidelity of exonuclease-deficient Pol epsilon is severalfold lower than that of proofreading-deficient forms of other replicative polymerases. Moreover the spectrum of errors shows a feature not seen with other A, B, C, or X family polymerases: a high proportion of transversions resulting from T.dTTP, T.dCTP, and C.dTTP mispairs. This unique error specificity and amino acid sequence alignments suggest that the structure of the polymerase active site of Pol epsilon differs from those of other B family members. We observed both similarities and differences between the spectrum of substitutions generated by proofreading-deficient Pol epsilon in vitro and substitutions occurring in vivo in a yeast strain defective in Pol epsilon proofreading and DNA mismatch repair. We discuss the implications of these findings for the role of Pol epsilon polymerase activity in DNA replication.

Amino Acid Sequence↗

PubMed2003

Evidence for preferential mismatch repair of lagging strand DNA replication errors in yeast.

Duplex DNA is replicated in the 5'-3' direction by coordinated copying of leading and lagging strand templates with somewhat different proteins and mechanics, providing the potential for differences in the fidelity of replication of the two strands. We previously showed that in Saccharomyces cerevisiae, active replication origins establish a strand bias in the rate of base substitutions resulting from replication of unrepaired 8-oxo-guanine (GO) in DNA. Lower mutagenesis was associated with replicating lagging strand templates. Here, we test the hypothesis that this bias is due to more efficient repair of lagging stand mismatches by measuring mutation rates in ogg1 strains with a reporter allele in two orientations at loci on opposite sides of a replication origin on chromosome III. We compare a MMR-proficient strain to strains deleted for the MMR genes MSH2, MSH6, MLH1, or EXOI. Loss of MMR reduces the strand bias by preferentially increasing mutagenesis for lagging strand replication. We conclude that GO-A mismatches generated during lagging strand replication are more efficiently repaired. This is consistent with the hypothesis that 5' ends of Okazaki fragments and PCNA, present at high density during lagging strand replication, are used as strand discrimination signals for mismatch repair in vivo.

Base Pair Mismatch↗

PubMed2003

Functions of human DNA polymerases eta, kappa and iota suggested by their properties, including fidelity with undamaged DNA templates.

Human DNA polymerases eta, kappa and iota are template-dependent, Y-family DNA polymerases that have been implicated in translesion DNA synthesis (TLS) in human cells. Here, we briefly review evidence that these exonuclease-deficient polymerases copy undamaged DNA with very low fidelity and unusual error specificity. Based on the base substitution specificity and other biochemical properties of DNA polymerases eta and iota, we consider the possibility that they participate in specialized DNA transactions that repair damaged DNA and/or generate mutations in the variable regions of immunoglobulin genes.

DNA↗

PubMed2003

Theoretical analysis of mutation hotspots and their DNA sequence context specificity.

Mutation frequencies vary significantly along nucleotide sequences such that mutations often concentrate at certain positions called hotspots. Mutation hotspots in DNA reflect intrinsic properties of the mutation process, such as sequence specificity, that manifests itself at the level of interaction between mutagens, DNA, and the action of the repair and replication machineries. The hotspots might also reflect structural and functional features of the respective DNA sequences. When mutations in a gene are identified using a particular experimental system, resulting hotspots could reflect the properties of the gene product and the mutant selection scheme. Analysis of the nucleotide sequence context of hotspots can provide information on the molecular mechanisms of mutagenesis. However, the determinants of mutation frequency and specificity are complex, and there are many analytical methods for their study. Here we review computational approaches for analyzing mutation spectra (distribution of mutations along the target genes) that include many mutable (detectable) positions. The following methods are reviewed: derivation of a consensus sequence, application of regression approaches to correlate nucleotide sequence features with mutation frequency, mutation hotspot prediction, analysis of oligonucleotide composition of regions containing mutations, pairwise comparison of mutation spectra, analysis of multiple spectra, and analysis of "context-free" characteristics. The advantages and pitfalls of these methods are discussed and illustrated by examples from the literature. The most reliable analyses were obtained when several methods were combined and information from theoretical analysis and experimental observations was considered simultaneously. Simple, robust approaches should be used with small samples of mutations, whereas combinations of simple and complex approaches may be required for large samples. We discuss several well-documented studies where analysis of mutation spectra has substantially contributed to the current understanding of molecular mechanisms of mutagenesis. The nucleotide sequence context of mutational hotspots is a fingerprint of interactions between DNA and DNA repair, replication, and modification enzymes, and the analysis of hotspot context provides evidence of such interactions.

Animals↗

PubMed2003

Computational analysis of mutation spectra.

Mutation frequencies vary along a nucleotide sequence, and nucleotide positions with an exceptionally high mutation frequency are called hotspots. Mutation hotspots in DNA often reflect intrinsic properties of the mutation process, such as the specificity with which mutagens interact with nucleic acids and the sequence-specificity of DNA repair/replication enzymes. They might also reflect structural and functional features of target protein or RNA sequences in which they occur. The determinants of mutation frequency and specificity are complex and there are many analytical methods for their study. This paper discusses computational approaches to analysing mutation spectra (distribution of mutations along the target genes) that include many detectable (mutable) positions. The following methods are reviewed: mutation hotspot prediction; pairwise and multiple comparisons of mutation spectra; derivation of a consensus sequence; and analysis of correlation between nucleotide sequence features and mutation spectra. Spectra of spontaneous and induced mutations are used for illustration of the complexities and pitfalls of such analyses. In general, the DNA sequence context of mutation hotspots is a fingerprint of interactions between DNA and DNA repair/replication/modification enzymes, and the analysis of hotspot context provides evidence of such interactions.

Amino Acid Motifs↗