Browsing by Author "Petes, Thomas Douglas"

Mitotic genome instability can occur during the repair of double-strand breaks (DSBs) in DNA, which arise from endogenous and exogenous sources. Studying the mechanisms of DNA repair in the budding yeast, Saccharomyces cerevisiae has shown that Homologous Recombination (HR) is a vital repair mechanism for DSBs. HR can result in a crossover event, in which the broken molecule reciprocally exchanges information with a homologous repair template. The current model of double-strand break repair (DSBR) also allows for a tract of information to non-reciprocally transfer from the template molecule to the broken molecule. These “gene conversion” events can vary in size and can occur in conjunction with a crossover event or in isolation. The frequency and size of gene conversions in isolation and gene conversions associated with crossing over has been a source of debate due to the variation in systems used to detect gene conversions and the context in which the gene conversions are measured.

In Chapter 2, I use an unbiased system that measures the frequency and size of gene conversion events, as well as the association of gene conversion events with crossing over between homologs in diploid yeast. We show mitotic gene conversions occur at a rate of 1.3x10-6 per cell division, are either large (median 54.0kb) or small (median 6.4kb), and are associated with crossing over 43% of the time.

DSBs can arise from endogenous cellular processes such as replication and transcription. Two important RNA/DNA hybrids are involved in replication and transcription: R-loops, which form when an RNA transcript base pairs with the DNA template and displaces the non-template DNA strand, and ribonucleotides embedded into DNA (rNMPs), which arise when replicative polymerase errors insert ribonucleotide instead of deoxyribonucleotide triphosphates. RNaseH1 (encoded by RNH1) and RNaseH2 (whose catalytic subunit is encoded by RNH201) both recognize and degrade the RNA in within R-loops while RNaseH2 alone recognizes, nicks, and initiates removal of rNMPs embedded into DNA. Due to their redundant abilities to act on RNA:DNA hybrids, aberrant removal of rNMPs from DNA has been thought to lead to genome instability in an rnh201Δ background.

In Chapter 3, I characterize (1) non-selective genome-wide homologous recombination events and (2) crossing over on chromosome IV in mutants defective in RNaseH1, RNaseH2, or RNaseH1 and RNaseH2. Using a mutant DNA polymerase that incorporates 4-fold fewer rNMPs than wild type, I demonstrate that the primary recombinogenic lesion in the RNaseH2-defective genome is not rNMPs, but rather R-loops. This work suggests different in-vivo roles for RNaseH1 and RNaseH2 in resolving R-loops in yeast and is consistent with R-loops, not rNMPs, being the the likely source of pathology in Aicardi-Goutières Syndrome patients defective in RNaseH2.

Telomeric sequences are often located internally on the chromosome in addition to their usual positions at the ends of the chromosome. These internally-located telomeric sequences have been termed “interstitial telomeric sequences” (ITSs). In humans, ITSs are non-randomly associated with translocation breakpoints in tumor cells and with chromosome fragile sites (regions of the chromosome that break in response to perturbed DNA replication). We previously showed that ITSs in yeast stimulated point mutations in DNA sequences adjacent to the ITS as well as several types of chromosomal rearrangements. The major class of these rearrangements was the terminal inversion, which inverted the chromosome segment between the ITS and the “true” chromosome telomere. In the current study, we examined the genetic control of these events. We show that the terminal inversions likely occur by the formation of a double-stranded DNA break within the ITS, followed by repair of the break utilizing the single-strand annealing pathway. The point mutations induced by the ITS require the error-prone DNA polymerase zeta. Unlike the terminal inversions, these events are not initiated by a double-stranded DNA break, but likely result from error-prone repair of a single-stranded DNA gap or recruitment of DNA polymerase zeta in the absence of DNA damage.

Double-stranded DNA breaks are potentially lethal lesions that can be repaired in mitotic cells by either homologous recombination (HR) or non-homologous end- joining (NHEJ) pathways. In the HR pathway, the broken DNA molecule is repaired using either the sister chromatid or the homolog as a template. Mitotic recombination events involving the homolog often result in loss of heterozygosity (LOH) of markers located distal to the crossover. In humans that are heterozygous for a mutation in a tumor suppressor gene, mitotic recombination leading to LOH can be an early step in cancer development.

In my thesis research, I analyzed mitotic recombination in the yeast Saccharomyces cerevisiae using oligonucleotide-containing microarrays to detect LOH of single-nucleotide polymorphisms (SNPs). In analyzing cells treated with ionizing radiation, I performed the first whole-genome analysis of LOH events done in any organism (Chapter 2). I showed that irradiated cells had between two and three unselected LOH events. I also showed that crossovers were often associated with non- reciprocal exchanges of genetic information (gene conversion events) and that these conversion events were more complex than predicted by standard models of homologous recombination.

In Chapter 3, I describe my mapping of spontaneous crossovers in a 1.1 Mb region of yeast chromosome IV. This analysis is the first high-resolution mitotic recombination map of a substantial fraction (about 10%) of a eukaryotic genome. I demonstrated the existence of recombination "hotspots" and showed that some of these hotspots were homolog-specific. Two of the strongest hotspots were formed by closely- spaced inverted repeats of retrotransposons. I demonstrated that the hotspot activity was a consequence of a secondary DNA structure formed by these repeats. Additionally, the majority of spontaneous LOH events reflect DNA lesions induced in unreplicated chromosomes during G1 of the cell cycle, indicating that G1-initiated lesions threaten genome stability more than G2-initiated lesions.

In Chapter 4, I describe mitotic crossovers associated with DNA replication stress induced by hydroxyurea (HU) treatment. Surprisingly, most HU-induced crossovers had conversion tracts indicative of DNA lesions initiated in G1. Additionally, HU- induced recombination events were very significantly associated with solo delta elements, a 330 bp sequence that is repeated several hundred times in the yeast genome.

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.

The genomes of many strains of baker’s yeast, Saccharomyces cerevisiae, contain multiple repeats of the copper-binding protein Cup1. Cup1 is a member of the metallothionein family, and is found in a tandem array on chromosome VIII. In this thesis, I describe studies that characterized these tandem arrays and their mechanism of formation across diverse strains of yeast. I show that CUP1 arrays are an illuminating model system for observing recombination in eukaryotes, and describe insights derived from these observations.

In our first study, we analyzed 101 natural isolates of S. cerevisiae in order to examine the diversity of CUP1-containing repeats across different strains. We identified five distinct classes of repeats that contain CUP1. We also showed that some strains have only a single copy of CUP1. By comparing the sequences of all the strains, we were able to elucidate the mechanism of formation of the CUP1 tandem arrays, which involved unequal non-homologous recombination events starting from a strain that had only a single CUP1 gene. Our observation of CUP1 repeat formation allows more general insights about the formation of tandem repeats from single-copy genes in eukaryotes, which is one of the most important mechanisms by which organisms evolve.

In our second study, we delved deeper into our mechanistic investigations by measuring the relative rates of inter-homolog and intra-/inter-sister chromatid recombination in CUP1 tandem arrays. We used a diploid strain that is heterozygous both for insertion of a selectable marker (URA3) inside the tandem array, and also for markers at either end of the array. The intra-/inter-sister chromatid recombination rate turned out to be more than ten-fold greater than the inter-homolog rate. Moreover, we found that loss of the proteins Rad51 and Rad52, which are required for most inter-homolog recombination, did not greatly reduce recombination in the CUP1 tandem repeats. Additionally, we investigated the effects of elevated copper levels on the rate of each type of recombination at the CUP1 locus. Both types of recombination are increased at high concentrations of copper (as is known to be the case for CUP1 transcription). Furthermore, the inter-homolog recombination rate at the CUP1 locus is higher than the average over the genome during mitosis, but is lower than the average during meiosis.

The research described in Chapter 2 is published in 2014.

Cancer cells often have elevated frequencies of chromosomal aberrations, and it is likely that loss of genome stability is one driving force behind tumorigenesis. Deficiencies in DNA replication, DNA repair, or cell cycle checkpoints can all contribute to increased rates of chromosomal duplications, deletions and translocations. The human ATM and ATR proteins are known to participate in the DNA damage response and DNA replication checkpoint pathways and are critical to maintaining genome stability. The Saccharomyces cerevisiae homologues of ATM and ATR are Tel1p and Mec1p, respectively. Because Tel1p and Mec1p are partially functionally redundant, loss of both Tel1p and Mec1p in haploid yeast cells (tel1 mec1 strains) results in synergistically elevated rates of chromosomal aberrations, including terminal duplications, chromosomal duplications, and telomere-telomere fusions. To determine the effect of Tel1p and Mec1p on chromosome aberrations that cannot be recovered in haploid strains, such as chromosome loss, I investigated the phenotypes associated with the tel1 mec1 mutations in diploid cells. In the absence of induced DNA damage, tel1 mec1 diploid yeast strains exhibit extremely high rates of aneuploidy and chromosome rearrangements. There is a significant bias towards trisomy of chromosomes II, VIII, X, and XII, whereas the smallest chromosomes I and VI are commonly monosomic.

The telomere defects associated with tel1 mec1 strains do not cause the high rates of aneuploidy, as restoring wild-type telomere length in these strains by expression of the Cdc13p-Est2p fusion protein does not prevent cells from becoming aneuploid. The tel1 mec1 diploids are not sensitive to the microtubule-destabilizing drug benomyl, nor do they arrest the cell cycle in response to the drug, indicating that the spindle assembly checkpoint is functional. The chromosome missegregation phenotypes of tel1 mec1 diploids mimic those observed in mutant strains that do not achieve biorientation of sister chromatids during mitosis.

The chromosome rearrangements in tel1 mec1 cells reflect both homologous recombination between non-allelic Ty elements, as well as non-homologous end joining (NHEJ) events. Restoring wild-type telomere length with the Cdc13p-Est2p fusion protein substantially reduces the levels of chromosome rearrangements (terminal additions and deletions of chromosome arms, interstitial duplications, and translocations). This result suggests that most of the rearrangements in tel1 mec1 diploids are initiated by telomere-telomere fusions. One common chromosome rearrangement in tel1 mec1 strains is an amplification of sequences on chromosome XII between the left telomere and rDNA sequences on the right arm. I have termed this aberration a "schromosome." Preliminary evidence indicates that the schromosome exists in the tel1 mec1 cells as an uncapped chromosome fragment that gets resected over time.

A mitotically dividing cell regularly experiences DNA damage including double-stranded DNA breaks (DSBs). Homologous mitotic recombination is an important mechanism for the repair of DSBs, but inappropriate repair of DNA breaks can lead to genome instability. Despite more than 70 years of research, the mechanism of mitotic recombination is still not understood. By genetic and physical studies in the yeast Saccharomyces cerevisiae, I investigated the mechanism of reciprocal mitotic crossovers. Since spontaneous mitotic recombination events are very infrequent, I used a diploid strain that allowed for selection of cells that had the recombinant chromosomes expected for a reciprocal crossover (RCO). The diploid was also heterozygous for many single-nucleotide polymorphisms, allowing the accurate mapping of the recombination events.

I mapped spontaneous crossovers to a resolution of about 4 kb in a 120 kb region of chromosome V. This analysis is the first large-scale mapping of mitotic events performed in any organism. One region of elevated recombination was detected (a "hotspot") and the region near the centromere of chromosome V had low levels of recombination ("coldspot"). This analysis also demonstrated the crossovers were often associated with the non-reciprocal transfer of information between homologous chromosomes; such events are termed "gene conversions" and have been characterized in detail in the products of meiotic recombination. The amount of DNA transferred during mitotic gene conversion events was much greater than that observed for meiotic conversions, 12 kb and 2 kb, respectively. In addition, about 40% of the conversion events had patterns of marker segregation that are most simply explained as reflecting the repair of a chromosome that was broken in G1 of the cell cycle.

To confirm this unexpected conclusion, I examined the crossovers and gene conversion events induced by gamma irradiation in G1- and G2-arrested diploid yeast cells. The gene conversion patterns of G1-irradiated cells (but not G2-irradiated cells) mimic the conversion events associated with spontaneous reciprocal crossovers (RCOs), confirming my hypothesis that many spontaneous crossovers are initiated by a DSB on an unreplicated chromosome. In conclusion, my results have resulted in a new understanding of the properties of mitotic recombination within the context of cell cycle.

Topoisomerase 1 (Top1), a Type IB topoisomerase, functions to relieve transcription- and replication-associated torsional stress in DNA. Top1 cleaves one strand of DNA, covalently associates with the 3’ end of the nick to form a Top1-cleavage complex (Top1cc), passes the intact strand through the nick and finally re-ligates the broken strand. The chemotherapeutic drug, Camptothecin, intercalates at a Top1cc and prevents the crucial re-ligation reaction that is mediated by Top1, resulting in the conversion of a nick to a toxic double-strand break during DNA replication or the accumulation of Top1cc. This mechanism of action preferentially targets rapidly dividing tumor cells, but can also affect non-tumor cells when patients undergo treatment. Additionally, Top1 is found to be elevated in numerous tumor tissues making it an attractive target for anticancer therapies. We investigated the effects of Top1 on genome stability, effects of persistent Top1-cleavage complexes and elevated Top1 levels, in Saccharomyces cerevisiae. We found that increased levels of the Top1cc resulted in a five- to ten-fold increase in reciprocal crossovers, three- to fifteen fold increase in mutagenesis and greatly increased instability within the rDNA and CUP1 tandem arrays. Increased Top1 levels resulted in a fifteen- to twenty-two fold increase in mutagenesis and increased instability in rDNA locus. These results have important implications for understanding the effects of CPT and elevated Top1 levels as a chemotherapeutic agent.