Sensitized genetic systems and DNA at-risk motif reporters have been developed during several years investigating genetic risks and synergistic interactions with DNA metabolic defects. These two genetic approaches have allowed us to identify situations resulting in high genome instability and to understand molecular mechanisms underlying the instability. Recently we combined genetic methods with physical observation of DNA repair and chromosome dynamics.? Repeat motifs have been identified in many organisms that are at high risk for genetic change in wild-type or mutation-prone cells. They can be a source genomic instability, cancer and in some cases provide changes that extend the host range of infectious microbes. We surmised from our various experimental approaches that many of these At Risk Motifs (ARMs) can form non-canonical DNA structures that are poor substrates for replication or post-replication repair. This led us to propose that ARMs can be a major source of genome instability. Once the underlying mechanisms are unraveled, ARMs also provide a powerful tool of discovery in mechanistic studies of DNA metabolism. Using a variety of genetic and molecular approaches with yeast, we have characterized several types of ARMs and employed them to address mechanisms of genome instability. These include small direct repeats separated by up to 100 bases, a motif common in all organisms, long inverted repeats in configurations similar to many arrangements of the frequently occurring Alu and LINE sequences in the human genome, homonucleotide runs as short as 8 to 10 bases that can lead to extremely high levels of frameshift mutation and other types of unstable mini- and microsatellites. We have established that defects in several aspects of DNA metabolic processes greatly exacerbate the instability associated with ARMs, in essence dramatically increasing the risk associated with ARMs.? Recently we addressed another set of challenges coming from transient single-strand DNA (ssDNA) state that increases genome instability. ssDNA can form during the repair of DSBs, replication fork progression, abnormal processing of telomeres and other DNA metabolic reactions. Since various major DNA repair mechanisms function specifically on double-strand DNA (dsDNA), ssDNA would be a poor substrate for DNA repair and thus result in increased risk to genome maintenance and stability.? DNA repair, replication and processing of DNA intermediates require coordinated interactions between many proteins. The combination of subtle changes in one or more DNA metabolic acting at ARMs can lead to synergistic increases in genome instability. We employ a variety of sophisticated genetic and biochemical approaches to identify genes that are important for maintaining genome stability. Our studies have concentrated on the interplay between genes involved in DNA replication, double-strand break repair, mismatch repair and base excision repair in maintaining genome stability, particularly at ARM sites and transient ssDNA regions.? Recently we directed research to understanding the special roles of DNA replication machinery in telomeres. We used genetic interaction with defects in pif1-antitelomerase and physical measurements of the level of homeostasis in the size of telomeric repeats as tools to highlight telomere-related mechanisms.? 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 pif1 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. In summary our data highlight the importance of the lagging strand functions in telomere replication and homeostasis.? DNA replication components are also 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 single strand breaks (SSBs) occur at AP sites if chromosomal DNA is exposed to high temperature (55oC) during sample preparation for PFGE while few breaks are formed if the DNA is processed at 30o C. If closely spaced on a chromosome, SSBs can give rise to secondary chromosomal DSBs that are detectable by PFGE. We established the induction of heat labile DSBS following MMS treatment. The heat-labile sites were efficiently repaired after incubating G1 cells for 24 hr in buffer. Consistent with a proposed role for BER in repair of heat-labile sites, the repair was blocked during post-treatment incubation of a mutant deleted for the the glycosylase MAG1 gene. Simultaneous deletion of AP-endonucleases APN1 and APN2 led to high amount of DSBs detected even when sample was processed at 30oC, indicating that heat-labile sites can be converted into breaks by other BER enzymes. These DSBs were suppressed by a mag1-deletion suggesting DSB formation is the result of the downstream in vivo processing of AP-sites. Thus, our assay provides for the identification of novel factors involved in BER. In combination with other physical methods monitoring repair, such as Q-PCR we plan to specifically address repair of closely spaced ssDNA damages.? In order to address the risks arising from transient formation of 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. After up to 24 kb of ssDNA has been generated on either side of the break, ssDNA oligonucleotide is supplied to repair a DSB. Our results led us to conclude that in the course of this repair large regions of ssDNA are restored to the dsDNA of the intact chromosome. This observation sets the stage for addressing the questions about the risk presented by transiently formed large regions of ssDNA. Currently we are exploring the role of environmental DNA damage in such regions.
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