Browsing by Author "Ayyar, Sandeep"

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    Break-Induced Replication and Genome Stability
    (MDPI, 2012-10-16) Sakofsky, Cynthia J.; Ayyar, Sandeep; Malkova, Anna; Biology, School of Science
    Genetic instabilities, including mutations and chromosomal rearrangements, lead to cancer and other diseases in humans and play an important role in evolution. A frequent cause of genetic instabilities is double-strand DNA breaks (DSBs), which may arise from a wide range of exogeneous and endogeneous cellular factors. Although the repair of DSBs is required, some repair pathways are dangerous because they may destabilize the genome. One such pathway, break-induced replication (BIR), is the mechanism for repairing DSBs that possesses only one repairable end. This situation commonly arises as a result of eroded telomeres or collapsed replication forks. Although BIR plays a positive role in repairing DSBs, it can alternatively be a dangerous source of several types of genetic instabilities, including loss of heterozygosity, telomere maintenance in the absence of telomerase, and non-reciprocal translocations. Also, mutation rates in BIR are about 1000 times higher as compared to normal DNA replication. In addition, micro-homology-mediated BIR (MMBIR), which is a mechanism related to BIR, can generate copy-number variations (CNVs) as well as various complex chromosomal rearrangements. Overall, activation of BIR may contribute to genomic destabilization resulting in substantial biological consequences including those affecting human health.
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    The Molecular Mechanism of Break Induced Replication
    (2013-02-14) Ayyar, Sandeep; Malkova, Anna
    DNA double strand break (DSB) is one of the most threatening of all types of DNA damages as it leads to a complete breakage of the chromosome. The cell has evolved several mechanisms to repair DSBs, one of which is break-induced replication (BIR). BIR repair of DSBs occurs through invasion of one end of the broken chromosome into a homologous template followed by processive replication of DNA from the donor molecule. BIR is a key cellular process and is implicated in the restart of collapsed replication forks and several chromosomal instabilities. Recently, our lab demonstrated that the fidelity of DNA synthesis associated with BIR in yeast Saccharomyces Cerevisiae is extremely low. The level of frameshift mutations associated with BIR is 1000-fold higher as compared to normal DNA replication. This work demonstrates that BIR stimulates base substitution mutations, which comprise 90% of all point mutations, making them 400-1400 times more frequent than during S-phase DNA replication. We show that DNA Polymerase δ proofreading corrects many of the base substitutions in BIR. Further, we demonstrate that Pif1, a 5’-3’ DNA helicase, is responsible for making BIR efficient and also highly mutagenic. Pif1p is responsible for the majority of BIR mutagenesis not only close to the DSB site, where BIR is less stable but also at chromosomal regions far away from the DSB break site, where BIR is fast, processive and stable. This work further reveals that, at positions close to the DSB, BIR mutagenesis in the absence of Pif1 depends on Rev3, the catalytic subunit of translesion DNA Polymerase ζ. We observe that mutations promoted by Pol ζ are often complex and propose that they are generated by a Pol ζ- led template switching mechanism. These complex mutations were also found to be frequently associated with gross chromosomal rearrangements. Finally we demonstrate that BIR is carried out by unusual conservative mode of DNA synthesis. Based on this study, we speculate that the unusual mode of DNA synthesis associated with BIR leads to various kinds of genomic instability including mutations and chromosomal rearrangements.
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    PIF1 HELICASE AND POLYMERASE ZETA (ζ) CHARACTERIZE TWO PATHWAYS OF MUTAGENESIS ASSOCIATED WITH BREAK INDUCED REPLICATION.
    (Office of the Vice Chancellor for Research, 2012-04-13) Ayyar, Sandeep; Sakofsky, Cynthia; Deem, Angie; Malkova, Anna
    The fidelity of DNA synthesis differs among the various processes in which it is involved. While normal S-phase DNA replication is highly accu-rate, DNA synthesis associated with DNA repair is often error-prone. Recent-ly, we have analyzed the accuracy of Break-induced replication, which is a unique cellular process that mimics normal DNA replication in its processivity and rate, but is initiated at double-strand breaks (DSBs) rather than at repli-cation origins. We have demonstrated that BIR is associated with approxi-mately a thousand-fold increase of the rate of frameshift mutations as com-pared to spontaneous events. Here we have identified 5’ – 3’ helicase Pif1p and translesion polymerase Pol ζ as the two major components in promoting frameshift mutations associated with BIR. We have also employed a rever-sion assay using base substitution reporter ura 3-29 to demonstrate that BIR elevates base substitution mutations by a fold of 400 over normal DNA repli-cation. This mutagenic character led us to explore the mode of repair synthesis associated with BIR.Our data suggests that BIR maybe following an unusual, conservative mode of synthesis very different from the usual semiconserva-tive mode of synthesis followed by normal S-phase DNA replication.