Mechanisms Of Genome Instability

Environmental Health Sciences

Investigators

Linked publications, trials & patents

Abstract

REPLICATION FACTORS AFFECTING GENOME STABILITY:[unreadable] We have addressed the special roles that the DNA replication machinery plays in lagging strand maturation across the genome and in telomeres of budding yeast. To investigate telomere-related activities, genetic interactions were examined in association with defects in pif1-antitelomerase and these findings were correlated with physical measurements of homeostasis in the size of telomeric repeats. To understand the interplay of components involved in the genome-wide Okazaki maturation process, we employed a deletion allele of the gene controlling the nonessential subunit of DNA polymerase delta (POL32). The Rad27/Fen1 5'-flap endonuclease has been implicated in the maturation of Okazaki fragments during lagging strand replication in yeast based on its biochemical activities, mutation spectra, and genetic interactions. We found that both pif1 and pol32 mutations can modulate synthetic lethality in yeast resulting from a combination of a rad27-null or partial mutant (rad27-p, lacking interaction with PCNA) with other defects in lagging strand replication and genome maintenance. Importantly, we found that the pol32-null can serve as a suppressor of the synthetic lethality in a combination of the rad27-null defect with a mutation eliminating the 3' to 5' exonuclease activity associated with DNA polymerase delta. Overall, our findings have enabled us to dissect important functions in lagging strand maturation and to identify lagging strand functions in telomere replication and homeostasis.[unreadable] [unreadable] BER[unreadable] The components of DNA replication are involved in many kinds of DNA repair, including base excision repair (BER). BER is common to all cells and provides relief from a variety of lesions. While there is considerable information about BER mechanisms gained from in vitro studies, there is little understanding of cellular events. To address directly the in vivo components of BER, we developed a method to detect methylmethane-sulfonate (MMS) induced base damage and repair using pulsed field gel electrophoresis (PFGE) analysis of secondary DSBs that arise in full-length chromosome DNA molecules of budding yeast. Abasic sites (AP sites) in DNA formed at methylated bases are heat-labile, and these give rise to single strand breaks (SSBs) if chromosomal DNA is exposed to high temperature (55oC) during sample preparation for PFGE. If, instead, the DNA is processed at 30o C, few breaks are formed. When SSBs are closely spaced, they can give rise to secondary chromosomal DSBs that are detectable by PFGE. We established the induction of heat labile DSBs following MMS treatment of G1 haploid cells. The heat-labile sites were efficiently repaired after incubating cells for 24 hr in buffer. The capability for repair was examined in mutants considered to be important to BER, such as MAG1, APN1, APN2 and RAD27. This was followed by a search for additional genes that might play a role in BER using the system that we developed. We found that efficient BER requires the nonessential subunit of DNA polymerase delta, POL32. This also provides a proof of principle for the utility of our assay in identifying new components in BER.[unreadable] [unreadable] SINGLE-STRAND DNA [unreadable] There are several processes in the cell that might lead to the transient generation of ssDNA. In order to address possible risks to genome stability that might arise with ssDNA, we utilized our recently developed experimental system where a site-specific double-strand break (DSB) is generated followed by generation of ssDNA by end-resection see 1 Z01 ES065072-17 LMG. We had established that up to 24 kb of ssDNA can be generated on either side of a DSB based on the ability of externally supplied ssDNA oligonucleotide to interact with the region. Our results led us to conclude that in the course of repair of the region, the large regions of ssDNA are restored to the dsDNA state of the intact chromosome. Importantly, we have established that genes in the ssDNA region that is created after DSB induction are hypermutable with a 1000-fold increase over the spontaneous rate of 10-6 to 10-7 per cell per generation. Furthermore, the DNA damaging agents UV-C and methyl methanesulfonate enhanced this hypermutability up to 100-fold more. Nearly all the UV-induced mutations were identified with pyrimidines in the nonresected strand, demonstrating directly that ssDNA can be especially vulnerable to mutagenesis. In support of this, there was a striking multiplicity of mutations with up to six independent changes separated by hundreds of nucleotides after a nonlethal dose of UV-C (36 J/m2). These results establish that long transient ss DNA stretches can be restored to the ds state even when they contain multiple lesions. The greatly increased induced, as well as spontaneous, mutagenesis was largely attributable to the bypass DNA polymerase zeta controlled by REV3 (as well as REV1) while the post-replication repair genes RAD5 and UBC13 had little effect. Hypermutability also depended on PCNA-monoubiquitination based on findings with a pol30-K164R mutant. [unreadable] We conclude that ssDNA is highly prone to damage-induced hypermutability through a process that requires bypass during the DNA synthesis process that restores damaged long stretches of ssDNA to dsDNA. Remarkably, the level of hypermutability can reach levels comparable to somatic hypermutation in mammalian immunoglobulin genes.