The genomes of all organisms are subject to change including alterations in chromosome number, chromosome structure (translocations, deletions, duplications), or single-base-pair mutations (point mutations). Although the frequency of these changes is very low in wild-type cells, certain mutations (mutators) greatly elevate the rate of instability. Most solid tumors have very high rates of chromosome rearrangements. In this proposal, DNA microarrays and high-throughput DNA sequencing will be used to perform a genome-wide analysis of recombination events in strains of the yeast Saccharomyces cerevisae that are genetically unstable as a model for understanding cancers with high levels of chromosome rearrangements. In addition, the mapping of recombination events in yeast strains lacking topoisomerases will be relevant to understanding the possible consequences of the chemotherapeutic use of topoisomerase inhibitors. One rationale for this analysis is that regions that have high levels of mitotic recombination also have high levels of double-stranded DNA breaks (DSBs), and that mapping the positions of the recombination events maps the positions of these DSBs. Mitotic recombination events in diploid strains in which the two homologous chromosomes are not identical result in loss of heterozygosity (LOH). Diploid strains heterozygous for about 55,000 single-nucleotide-polymorphisms (SNPs) were constructed and will be analyzed using microarrays that can detect whether strains are heterozygous or homozygous for these polymorphisms (SNP arrays) to map LOH events. These events will be mapped throughout the genome in strains known to have very high levels of genetic instability including: 1) strains with low levels of alpha DNA polymerase, 2) tel1 mec1 sml1 strains (lacking homologues of the human ATM and ATR genes), and 3) strains with mutations in the topoisomerases Top1p and/or Top2p. These studies will define regions of the genome that are prone to DSB formation under these three different types of genome-destabilizing conditions. As a proof of principle, we mapped more than 200 LOH events in strains with 10-fold reduced levels of alpha DNA polymerase and showed that these events are non-randomly associated with DNA sequence motifs known to slow DNA replication forks. To complement this approach, microarrays will be used to map the locations of gamma-H2AX in strains with low alpha DNA polymerase or mutations in topoisomerase genes. Since gamma-H2AX is recruited to sites of DNA damage, the regions frequently associated with LOH are likely to co-localize with regions that have high levels of gamma-H2AX. High-throughput DNA sequencing of isolates of the same three types of strains described above will also be done to detect genetic alterations (single-base-pair changes and small insertions/deletions) that cannot be detected by SNP microarrays. Finally, we plan to map LOH events in mammalian cells that are exposed to topoisomerase inhibitors.
Genetic instability, resulting in high levels of chromosome rearrangements, is found in the cells of most solid tumors. We will use DNA microarrays and high-throughput DNA sequencing to perform a genome-wide analysis of strains of the yeast Saccharomyces cerevisae that have very high levels of chromosome rearrangements. Our mapping of yeast chromosome regions that are prone to rearrangements will help us understand the genetic instability associated with cancer.
|Zheng, Dao-Qiong; Zhang, Ke; Wu, Xue-Chang et al. (2016) Global analysis of genomic instability caused by DNA replication stress in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 113:E8114-E8121|
|Deng, Sarah K; Yin, Yi; Petes, Thomas D et al. (2015) Mre11-Sae2 and RPA Collaborate to Prevent Palindromic Gene Amplification. Mol Cell 60:500-8|
|O'Connell, Karen; Jinks-Robertson, Sue; Petes, Thomas D (2015) Elevated Genome-Wide Instability in Yeast Mutants Lacking RNase H Activity. Genetics 201:963-75|
|Clausen, Anders R; Lujan, Scott A; Burkholder, Adam B et al. (2015) Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation. Nat Struct Mol Biol 22:185-91|
|Chan, Kin; Roberts, Steven A; Klimczak, Leszek J et al. (2015) An APOBEC3A hypermutation signature is distinguishable from the signature of background mutagenesis by APOBEC3B in human cancers. Nat Genet 47:1067-72|
|Yin, Yi; Petes, Thomas D (2014) The role of Exo1p exonuclease in DNA end resection to generate gene conversion tracts in Saccharomyces cerevisiae. Genetics 197:1097-109|
|Anand, Ranjith P; Tsaponina, Olga; Greenwell, Patricia W et al. (2014) Chromosome rearrangements via template switching between diverged repeated sequences. Genes Dev 28:2394-406|
|Zhao, Ying; Strope, Pooja K; Kozmin, Stanislav G et al. (2014) Structures of naturally evolved CUP1 tandem arrays in yeast indicate that these arrays are generated by unequal nonhomologous recombination. G3 (Bethesda) 4:2259-69|
|Song, Wei; Dominska, Margaret; Greenwell, Patricia W et al. (2014) Genome-wide high-resolution mapping of chromosome fragile sites in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 111:E2210-8|
|Sakofsky, Cynthia J; Roberts, Steven A; Malc, Ewa et al. (2014) Break-induced replication is a source of mutation clusters underlying kataegis. Cell Rep 7:1640-8|
Showing the most recent 10 out of 22 publications