Browsing by Author "Yin, Yi"

In diploid yeast cells, mitotic recombination is very important for repairing double-strand breaks (DSB). When repair of a DSB results in crossovers, it may cause loss of heterozygosity (LOH) of markers centromere-distal to the DSB in both daughter cells. Gene conversion events unassociated with crossovers cause LOH for an interstitial section of a chromosome. Alternatively, DSBs can initiate break-induced replication (BIR), causing LOH in only one of the daughter cells. Mapping mitotic LOH contributes to understanding of mechanisms for repairing DSBs and distribution of these recombinogenic lesions. Methods for selecting mitotic crossovers and mapping the positions of crossovers have recently been developed in our lab. Our current approach uses a diploid yeast strain that is heterozygous for about 55,000 SNPs, and employs SNP-Microarrays to map LOH events throughout the genome. These methods allow us to examine selected crossovers on chromosome V and unselected mitotic recombination events (crossovers, gene conversion events unassociated with crossovers, and BIR events) at about 1 kb resolution across the genome.

Mitotic recombination can be greatly induced by UV radiation. However, prior to my research, the nature of the recombinogenic lesions and the distribution of UV-induced recombination events were relatively uncharacterized. Using SNP microarrays, we constructed maps of UV-induced LOH events in G1-synchronized cells. Mitotic crossovers were stimulated 1500-fold and 8500-fold by UV doses of 1 J/m2 and 15 J/m2, respectively, compared to spontaneous events. Additionally, cells treated with 15 J/m2 have about eight unselected LOH events per pair of sectors, including gene conversions associated and unassociated with crossovers as well as BIR events. These unselected LOH events are distributed randomly throughout the genome with no particular hotspots; however, the rDNA cluster was under-represented for the initiation of crossover and BIR events. Interestingly, we found that a high fraction of recombination events in cells treated with 15 J/m2 reflected repair of two sister chromatids broken at roughly the same position. In cells treated with 1 J/m2, most events reflect repair of a single broken sister chromatid (Chapter 2).

The primary pathway to remove pyrimidine dimers introduced by UV is the nucleotide excision repair (NER) pathway. In NER, the dimer is excised to generate a 30-nucleotide gap that can be replicated to form DSBs if not filled in before DNA replication. The NER gap can also be expanded by Exo1p to form single stranded gaps greater than one kilobase. Alternatively, in the absence of NER, unexcised dimers could result in blocks of DNA replication forks. Resolving the stalled replication fork could lead to recombinogenic breaks. In Chapter 3 and Chapter 4, we analyzed recombination events in strains defective in various steps of processing of UV-induced DNA damage, including exo1 and rad14 mutants.

In Chapter 3, I show that Exo1p-expanded NER gaps contribute to UV-induced recombination events. Interestingly, I also found that Exo1p is also required for the hotspot activity of a spontaneous crossover hotspot involving a pair of inverted Ty repeats. In addition to its role of expanding a nick to a long single-stranded gap, Exo1p is also a major player in DSB end resection. Therefore, I examined the gene conversion tract lengths in strains deleted for EXO1. I found that, although crossover-associated gene conversion tracts become shorter in the exo1 mutant as expected, noncrossover tract lengths remained unaffected. As a result, noncrossover tracts are longer than crossover tracts in the exo1 mutant while the opposite result was observed in the wild-type strains. I proposed models to rationalize this observation.

In Chapter 4, to investigate whether the substantial recombinogenic effect in UV in G1-synchronized cells requires NER, we mapped UV-induced LOH events in NER-deficient rad14 diploids treated with 1 J/m2. Mitotic recombination between homologs was greatly stimulated, which suggests that dimers themselves can also cause recombination without processing by NER. We further show that UV-induced inter-homolog recombination events (noncrossover, crossover and BIR) depend on the resolvase Mus81p, and are suppressed by Mms2p-mediated error-free post-replication repair pathway.

The research described in Chapters, 2, 3, and 4 are in the publications Yin and Petes (2013), Yin and Petes (2014), and Yin and Petes (2015), respectively.

During mitotic cell cycles, DNA experiences many types of endogenous and exogenous damaging agents that could potentially cause double strand breaks (DSB). In S. cerevisiae, DSBs are primarily repaired by mitotic recombination and as a result, could lead to loss-of-heterozygosity (LOH). Genetic recombination can happen in both meiosis and mitosis. While genome-wide distribution of meiotic recombination events has been intensively studied, mitotic recombination events have not been mapped unbiasedly throughout the genome until recently. Methods for selecting mitotic crossovers and mapping the positions of crossovers have recently been developed in our lab. Our current approach uses a diploid yeast strain that is heterozygous for about 55,000 SNPs, and employs SNP-Microarrays to map LOH events throughout the genome. These methods allow us to examine selected crossovers and unselected mitotic recombination events (crossover, noncrossover and BIR) at about 1 kb resolution across the genome. Using this method, we generated maps of spontaneous and UV-induced LOH events. In this study, we explore machine learning and variable selection techniques to build a predictive model for where the LOH events occur in the genome.

Randomly from the yeast genome, we simulated control tracts resembling the LOH tracts in terms of tract lengths and locations with respect to single-nucleotide-polymorphism positions. We then extracted roughly 1,100 features such as base compositions, histone modifications, presence of tandem repeats etc. and train classifiers to distinguish control tracts and LOH tracts. We found interesting features of good predictive values. We also found that with the current repertoire of features, the prediction is generally better for spontaneous LOH events than UV-induced LOH events.