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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM052319-16A1
Application #
8288987
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Janes, Daniel E
Project Start
1995-05-01
Project End
2016-03-31
Budget Start
2012-04-06
Budget End
2013-03-31
Support Year
16
Fiscal Year
2012
Total Cost
$384,133
Indirect Cost
$136,528
Name
Duke University
Department
Genetics
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
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
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
Lujan, Scott A; Clausen, Anders R; Clark, Alan B et al. (2014) Heterogeneous polymerase fidelity and mismatch repair bias genome variation and composition. Genome Res 24:1751-64
Tang, Wei; Dominska, Margaret; Gawel, Malgorzata et al. (2013) Genomic deletions and point mutations induced in Saccharomyces cerevisiae by the trinucleotide repeats (GAA·TTC) associated with Friedreich's ataxia. DNA Repair (Amst) 12:10-7
Tang, Wei; Dominska, Margaret; Greenwell, Patricia W et al. (2011) Friedreich's ataxia (GAA)n•(TTC)n repeats strongly stimulate mitotic crossovers in Saccharomyces cerevisae. PLoS Genet 7:e1001270
Kim, Hyun-Min; Narayanan, Vidhya; Mieczkowski, Piotr A et al. (2008) Chromosome fragility at GAA tracts in yeast depends on repeat orientation and requires mismatch repair. EMBO J 27:2896-906