Youri I. Pavlov

Somatic mutation hotspots correlate with DNA polymerase |[eta]| error spectrum

Mutational spectra analysis of 15 immunoglobulin genes suggested that consensus motifs RGYW and WA were universal descriptors of somatic hypermutation. Highly mutable sites, “hotspots”, that matched WA were preferentially found in one DNA strand and RGYW hotspots were found in both strands. Analysis of base-substitution hotspots in DNA polymerase error spectra showed that 33 of 36 hotspots in the human polymerase η spectrum conformed to the WA consensus. This and four other characteristics of polymerase η substitution specificity suggest that errors introduced by this enzyme during synthesis of the nontranscribed DNA strand in variable regions may contribute to strand-specific somatic hypermutagenesis of immunoglobulin genes at A-T base pairs.

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.

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.

Correlation of somatic hypermutation specificity and A-T base pair substitution errors by DNA polymerase η during copying of a mouse immunoglobulin κ light chain transgene

To test the hypothesis that inaccurate DNA synthesis by mammalian DNA polymerase η (pol η) contributes to somatic hypermutation (SHM) of Ig genes, we measured the error specificity of mouse pol η during synthesis of each strand of a mouse Ig κ light chain transgene. We then compared the results to the base substitution specificity of SHM of this same gene in the mouse. The in vitro and in vivo base substitution spectra shared a number of common features. A highly significant correlation was observed for overall substitutions at A-T pairs but not for substitutions at G-C pairs. Sixteen mutational hotspots at A-T pairs observed in vivo were also found in spectra generated by mouse pol η in vitro. The correlation was strongest for errors made by pol η during synthesis of the non-transcribed strand, but it was also observed for synthesis of the transcribed strand. These facts, and the distribution of substitutions generated in vivo, support the hypothesis that pol η contributes to SHM of Ig genes at A-T pairs via short patches of low fidelity DNA synthesis of both strands, but with a preference for the non-transcribed strand.

Functions of human DNA polymerases η, κ and ι suggested by their properties, including fidelity with undamaged DNA templates

Abstract Human DNA polymerases η, κ and ι 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 η and ι, 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.

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.

Mutator effects of overproducing DNA polymerase η (Rad30) and its catalytically inactive variant in yeast

DNA polymerase eta synthesizes DNA in vitro with low fidelity. Based on this, here we report the effects of deletion or increased expression of yeast RAD30 gene, encoding for polymerase eta (Pol eta), on spontaneous mutagenesis in vivo. Deletion of RAD30 did not affect spontaneous mutagenesis. Overproduction of Rad30p was slightly mutagenic in a wild-type yeast strain and moderately mutagenic in strains with inactive 3-->5-exonuclease of DNA polymerase epsilon or DNA mismatch repair. These data suggest that excess Rad30p reduces replication fidelity in vivo and that the induced errors may be corrected by exonucleolytic proofreading and DNA mismatch repair. However, the magnitude of mutator effect (only up to 10-fold) suggests that the replication fork is protected from inaccurate synthesis by Pol eta in the absence of DNA damage. Overproduction of catalytically inactive Rad30p was also mutagenic, suggesting that much of the mutator effect results from indirect perturbation of replication rather than from direct misincorporation by Pol eta. Moreover, while excess wild-type Pol eta primarily induced base substitutions in the msh6 and pms1 strains, excess inactive Rad30p induced both base substitutions and frameshifts. This suggests that more than one mutagenic mechanism is operating when RAD30 is overexpressed.

YcbX and yiiM, two novel determinants for resistance of Escherichia coli to N‐hydroxylated base analogues

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 analogues, 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 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 sulphurases. 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 sulphuration. 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.

Hypersensitivity of Escherichia coli Δ(uvrB-bio) Mutants to 6-Hydroxylaminopurine and Other Base Analogs Is Due to a Defect in Molybdenum Cofactor Biosynthesis

We have shown previously that Escherichia coli and Salmonella enterica serovar Typhimurium strains carrying a deletion of the uvrB-bio region are hypersensitive to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and related base analogs. This sensitivity is not due to the uvrB excision repair defect associated with this deletion because a uvrB point mutation or a uvrA deficiency does not cause hypersensitivity. In the present work, we have investigated which gene(s) within the deleted region may be responsible for this effect. Using independent approaches, we isolated both a point mutation and a transposon insertion in the moeA gene, which is located in the region covered by the deletion, that conferred HAP sensitivity equal to that conferred by the uvrB-bio deletion. The moeAB operon provides one of a large number of genes responsible for biosynthesis of the molybdenum cofactor. Defects in other genes in the same pathway, such as moa or mod, also lead to the same HAP-hypersensitive phenotype. We propose that the molybdenum cofactor is required as a cofactor for an as yet unidentified enzyme (or enzymes) that acts to inactivate HAP and other related compounds.

Base analog N6-hydroxylaminopurine mutagenesis in Escherichia coli: genetic control and molecular specificity.

We have studied the molecular specificity of the base analog N6-hydroxylaminopurine (HAP) in the E. coli lacI gene, as well as the effects of mutations in DNA repair and replication genes on HAP mutagenesis. HAP induced base substitutions of the two transition types (A . T-->G . C and G . C-->A . T) at equal frequency. This bi-directional transition specificity is consistent with in vitro primer extension experiments with the Klenow fragment of DNA polymerase I in which we observed that either dTTP or dCTP were incorporated opposite HAP in an oligonucleotide template. The spectrum of HAP-induced transitions was different from the spontaneous transitions in either a wild-type or a mismatch-repair-defective (mutL) strain. Mutations in genes controlling excision repair, exonucleolytic proofreading, mismatch correction, error-prone (SOS) repair and 8-oxo-guanine repair did not affect HAP-induced mutagenesis substantially. However, an extensive deletion of several genes in the uvrB-bio region conferred supersensitivity to the lethal and mutagenic effects of HAP, perhaps due to an effect on HAP metabolism. dnaE antimutator alleles reduced HAP-forward mutagenicity in allele-specific manner: dnaE911 reduced it several fold, while dnaE915 abolished it almost completely. The results obtained are consistent with the idea that HAP is mutagenic in E. coli via a pathway generating replication errors.