Altered Ig Hypermutation Pattern and Frequency in Complementary Mouse Models of DNA Polymerase zeta Activity

Somatic Hypermutation Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
The Journal of Immunology (Impact Factor: 5.36). 04/2012; 188(11):5528-37. DOI: 10.4049/jimmunol.1102629
Source: PubMed

ABSTRACT To test the hypothesis that DNA polymerase ζ participates in Ig hypermutation, we generated two mouse models of Pol ζ function: a B cell-specific conditional knockout and a knock-in strain with a Pol ζ mutagenesis-enhancing mutation. Pol ζ-deficient B cells had a reduction in mutation frequency at Ig loci in the spleen and in Peyer's patches, whereas knock-in mice with a mutagenic Pol ζ displayed a marked increase in mutation frequency in Peyer's patches, revealing a pattern that was similar to mutations in yeast strains with a homologous mutation in the gene encoding the catalytic subunit of Pol ζ. Combined, these data are best explained by a direct role for DNA polymerase ζ in Ig hypermutation.

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    ABSTRACT: Activation-induced cytidine deaminase (AID) is essential for anti-body diversification, namely somatic hypermutation (SHM) and class switch recombination (CSR). The deficiency of apurinic/ apyrimidinic endonuclease 1 (Ape1) in CH12F3-2A B cells reduces CSR to ∼20% of wild-type cells, whereas the effect of APE1 loss on SHM has not been examined. Here we show that, although APE1's endonuclease activity is important for CSR, it is dispensable for SHM as well as IgH/c-myc translocation. Importantly, APE1 defi-ciency did not show any defect in AID-induced S-region break formation, but blocked both the recruitment of repair protein Ku80 to the S region and the synapse formation between Sμ and Sα. Knockdown of end-processing factors such as meiotic re-combination 11 homolog (MRE11) and carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) further reduced the remaining CSR in Ape1-null CH12F3-2A cells. Together, our results show that APE1 is dispensable for SHM and AID-induced DNA breaks and may function as a DNA end-processing enzyme to fa-cilitate the joining of broken ends during CSR. class switch recombination | somatic hypermutation | DNA cleavage | DNA synapse formation | end processing U pon encountering antigens in the periphery, mature B cells undergo two types of genetic alterations, somatic hyper-mutation (SHM) and class switch recombination (CSR), in the Ig gene during the G1 phase (1, 2). Although mechanistically different, both events are initiated by the activation-induced cytidine de-aminase (AID) (3–5), which introduces single-strand DNA break (SSB) in the target DNA, namely the V region for SHM and the S region for CSR (6–9). Most SHMs are introduced during the repair of AID-induced SSBs through error-prone DNA synthesis by translesion polymerases, such as Polη and Polζ (10, 11). By contrast, efficient CSR requires a series of biological steps including conversion of AID-induced SSBs to double-strand breaks (DSBs), the synapsis formation of S-region broken ends, and their recombination (8, 12). Among them, processing of SSBs to DSBs without replication involves many enzymes in-cluding exonucleases, endonucleases, and/or helicases. Further-more, when the cleaved ends are blocked by unusual modified bases, they should be removed by end-processing enzymes such as carboxy-terminal binding protein (CtBP)-interacting protein (CtIP) and the MRN complex (13). Subsequently, the DSBs at the donor and acceptor S regions are joined by either the non-homologous end joining (NHEJ) or alternative end joining (A-EJ) pathway (14–17). During the NHEJ pathway that requires DSBs with blunt end or short overhang, additional end pro-cessing could occur at several steps before the recruitment of repair protein Ku80, or after synapse formation (13). The A-EJ pathway is used for the joining of DSB with long overhangs and also is involved in aberrant chromosomal translocations, which depend on uracil DNA glycosylase (UNG) (18). Both SHM and CSR take place during the G1 phase and are transcription-dependent (19). Because AID is known to in-troduce DNA cleavage in a limited number of non-Ig genes such as oncogene c-myc, AID targets are restricted (20). There are only a small number of enzymes that can cause DNA damage (genotoxic activity) in a transcription-dependent manner. Topoisomerase 1 (Top1) was recently shown to be genotoxic in neural cells that do not replicate (21). During transcription, Top1 normally regu-lates the DNA helix by transient SSBs, covalent association with DNA, DNA rotation around the helix, and relegation. However, Top1 occasionally may be trapped as covalently bound to DNA in the repetitive sequences that can form non-B struc-tures (22). We proposed that AID suppresses Top1 protein synthesis by deaminating cytidine (C) in miRNAs (23, 24). The reduction in the Top1 protein facilitates the formation of non-B DNA structures in the V and S regions of the Ig locus when they are actively transcribed. Such unusual DNA structure causes irrevers-ible DNA cleavage by Top1 because this structure suppresses the ordinary rotation around the helix of DNA (20, 22–24). Conversely, AID was also proposed to directly deaminate DNA, generating G:U mismatches that are detected and cleaved either by the base excision repair (BER) pathway including UNG and apurinic/apyrimidinic endonuclease 1 (APE1) or by the mis-match repair (MMR) enzymes including MutS homolog 2/MutS homolog 6 (Msh2/Msh6) (9, 25, 26). It is presumed that APE1 nicks DNA at abasic sites after uracil (U) removal by UNG, cre-ating SSBs that, after processing, give rise to SHM, CSR, or ab-errant recombinations/translocations (9). APE1 is a multifunctional protein that orchestrates multiple activities in the cell: (i) endonuclease activity for DNA damage repair (27), (ii) the redox activity for the maintenance of tran-scription factors (28), and (iii) binding to negative Ca
    Proceedings of the National Academy of Sciences 11/2014; 111(48). DOI:10.1073/pnas.1420221111 · 9.81 Impact Factor
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    ABSTRACT: Multiple sequence changes that are simultaneously introduced in a single DNA transaction have a higher probability of altering gene function than do single base substitutions. DNA polymerase zeta (Pol ζ) has been shown to introduce such clustered mutations under specific selective and/or DNA damage-producing conditions. In this study, a forward mutation assay was used to determine the specificity of spontaneous mutations generated in Saccharomyces cerevisiae when either wild-type Pol ζ or a mutator Pol ζ variant (rev3-L979F) bypasses endogenous lesions. Mutagenesis in strains proficient for nucleotide excision repair (NER) was compared to mutagenesis in NER-deficient strains that retain unrepaired endogenous DNA lesions in the genome. Compared to NER-proficient strains, NER-deficient rad14Δ strains have elevated mutation rates that depend on Pol ζ. Rates are most strongly elevated for tandem base pair substitutions and clusters of multiple, closely spaced mutations. Both types of mutations depend on Pol ζ, but not on Pol η. Rates of each are further elevated in yeast strains bearing the rev3-979F allele. The results indicate that when Pol ζ performs mutagenic bypass of endogenous, helix-distorting lesions, it catalyzes a short track of processive, error-prone synthesis. We discuss the implications of this unique catalytic property of Pol ζ to its evolutionary conservation and possibly to multistage carcinogenesis.Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.
    Environmental and Molecular Mutagenesis 12/2012; 53(9). DOI:10.1002/em.21728 · 2.55 Impact Factor
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    ABSTRACT: About 2% of human genetic polymorphisms have been hypothesized to arise via multinucleotide mutations (MNMs), complex events that generate SNPs at multiple sites in a single generation. MNMs have the potential to accelerate the pace at which single genes evolve and to confound studies of demography and selection that assume all SNPs arise independently. In this paper, we examine clustered mutations that are segregating in a set of 1,092 human genomes, demonstrating that MNMs become enriched as large numbers of individuals are sampled. We leverage the size of the dataset to deduce new information about the allelic spectrum of MNMs, estimating the percentage of linked SNP pairs that were generated by simultaneous mutation as a function of the distance between the affected sites and showing that MNMs exhibit a high percentage of transversions relative to transitions. These findings are reproducible in data from multiple sequencing platforms. Among tandem mutations that occur simultaneously at adjacent sites, we find an especially skewed distribution of ancestral and derived dinucleotides, with $\textrm{GC}\to \textrm{AA}$, $\textrm{GA}\to \textrm{TT}$ and their reverse complements making up 36% of the total. These same mutations dominate the spectrum of tandem mutations produced by the upregulation of low-fidelity Polymerase $\zeta$ in mutator strains of S. cerevisiae that have impaired DNA excision repair machinery. This suggests that low-fidelity DNA replication by Pol $\zeta$ is at least partly responsible for the MNMs that are segregating in the human population, and that useful information about the biochemistry of MNM can be extracted from ordinary population genomic data. We incorporate our findings into a mathematical model of the multinucleotide mutation process that can be used to correct phylogenetic and population genetic methods for the presence of MNMs.
    Genome Research 12/2013; 24(9). DOI:10.1101/gr.170696.113 · 13.85 Impact Factor


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