Browsing by Subject "Genome stability"

DNA replication is an intricate process within eukaryotic cells that must be precisely executed to preserve genetic information. This process begins at multiple start sites, or origins of replication, along each chromosome which are selected, licensed, and activated through cell-cycle regulated steps. Powerful reconstitution studies have identified the proteins involved in these processes, but they do not fully recapitulate the nuclear environment. Within the nucleus, the genome is organized in a chromatin structure consisting of DNA and all associated factors. At origins of replication, local chromatin contributes to origin identity and activation, but the precise chromatin dynamics that occur at these sites during helicase activation and initial DNA unwinding have not been fully explored. Additionally, how these steps are regulated to ensure genomic stability remain unstudied within the context of chromatin.

To address these questions, I have developed a conditional system that removes polymerase α function to capture helicase activation at replication origins in the budding yeast. Under restrictive conditions, these cells (cdc17-ts-FRB) do not initiate replication. When allowed to recover, replication appears to initiate outside origins, necessitating a delay in G2/M phase to repair unreplicated gaps at origins. To investigate origin chromatin and helicase movement prior to replication, I used MNase chromatin profiling alongside ChIP-seq for various replication factors. Chromatin in a 1 kb region around early, efficient replication origins is disrupted under restrictive conditions. The active helicase unwinds DNA out to 1 kb from these origins and is likely the source of the chromatin disruption. I next used the cdc17-ts-FRB conditional system to investigate the regulation of helicase progression in the absence of replication. I first tested whether the intra-S-phase checkpoint had a role in stalling the helicase 1 kb from the origin. Though removing checkpoint activation distributed helicase movement and chromatin disruption to late, inefficient origins, it did not alter the distance the helicase progressed from the origin. Instead, the helicase stalls as it leaves the AT-rich origin region and encounters sequences with higher GC content. These results provide in vivo support for the recently proposed “dead man’s switch” model for decreased helicase processivity when uncoupled from replication.

Helicase activation and origin unwinding are essential steps during DNA replication that expose ssDNA and thus have the potential to cause genomic instability. My studies have captured origin chromatin dynamics caused by an active helicase unwinding DNA, and have contributed evidence that the helicase may be intrinsically less processive in the absence of leading strand synthesis. These results may have implications for the mechanisms underlying human diseases involving polymerase α, and contribute to our growing understanding of how the eukaryotic cell preserves the integrity of the genome.

The complete and faithful duplication of the genome is essential to ensure normal cell division and organismal development. Eukaryotic DNA replication is initiated at multiple sites termed origins of replication that are activated at different time through S phase. The replication timing program is regulated by the S-phase checkpoint, which signals and repairs replicative stress. Eukaryotic DNA is packaged with histones into chromatin, thus DNA-templated processes including replication are modulated by the local chromatin environment such as post-translational modifications (PTMs) of histones.

One such epigenetic mark, methylation of lysine 20 on histone H4 (H4K20), has been linked to chromatin compaction, transcription, DNA repair and DNA replication. H4K20 can be mono-, di- and tri-methylated. Monomethylation of H4K20 (H4K20me1) is mediated by the cell cycle-regulated histone methyltransferase PR-Set7 and subsequent di-/tri- methylation is catalyzed by Suv4-20. Prior studies have shown that PR-Set7 depletion in mammalian cells results in defective S phase progression and the accumulation of DNA damage, which may be partially attributed to defects in origin selection and activation. Meanwhile, overexpression of mammalian PR-Set7 recruits components of pre-Replication Complex (pre-RC) onto chromatin and licenses replication origins for re-replication. However, these studies were limited to only a handful of mammalian origins, and it remains unclear how PR-Set7 impacts the replication program on a genomic scale. Finally, the methylation substrates of PR-Set7 include both histone (H4K20) and non-histone targets, therefore it is necessary to directly test the role of H4K20 methylation in PR-Set7 regulated phenotypes.

I employed genetic, cytological, and genomic approaches to better understand the role of H4K20 methylation in regulating DNA replication and genome stability in Drosophila melanogaster cells. Depletion of Drosophila PR-Set7 by RNAi in cultured Kc167 cells led to an ATR-dependent cell cycle arrest with near 4N DNA content and the accumulation of DNA damage, indicating a defect in completing S phase. The cells were arrested at the second S phase following PR-Set7 downregulation, suggesting that it was an epigenetic effect that coupled to the dilution of histone modification over multiple cell cycles. To directly test the role of H4K20 methylation in regulating genome integrity, I collaborated with the Duronio Lab and observed spontaneous DNA damage on the imaginal wing discs of third instar mutant larvae that had an alanine substitution on H4K20 (H4K20A) thus unable to be methylated, confirming that H4K20 is a bona fide target of PR-Set7 in maintaining genome integrity.

One possible source of DNA damage due to loss of PR-Set7 is reduced origin activity. I used BrdU-seq to profile the genome-wide origin activation pattern. However, I found that deregulation of H4K20 methylation states by manipulating the H4K20 methyltransferases PR-Set7 and Suv4-20 had no impact on origin activation throughout the genome. I then mapped the genomic distribution of DNA damage upon PR-Set7 depletion. Surprisingly, ChIP-seq of the DNA damage marker γ-H2A.v located the DNA damage to late replicating euchromatic regions of the Drosophila genome, and the strength of γ-H2A.v signal was uniformly distributed and spanned the entire late replication domain, implying stochastic replication fork collapse within late replicating regions. Together these data suggest that PR-Set7-mediated monomethylation of H4K20 is critical for maintaining the genomic integrity of late replicating domains, presumably via stabilization of late replicating forks.

In addition to investigating the function of H4K20me, I also used immunofluorescence to characterize the cell cycle regulated chromatin loading of Mcm2-7 complex, the DNA helicase that licenses replication origins, using H4K20me1 level as a proxy for cell cycle stages. In parallel with chromatin spindown data by Powell et al. (Powell et al. 2015), we showed a continuous loading of Mcm2-7 during G1 and a progressive removal from chromatin through S phase.

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.

The DNA double-strand break (DSB) is one of the most toxic genomic lesions that can occur in any living cell. Failure to repair DSBs results in cell cycle arrest and ultimately programmed cell death, while improper repair can lead to profound alterations or loss of genomic information through translocations, inversions, deletions and other genomic aberrations. Although the molecular events required for the repair of double-strand breaks (DSB) have been well characterized, the role of epigenetic processes in the recognition and repair of DSBs has only been investigated at low resolution. I tested several site-specific DSB induction systems and found that that the HO endonuclease was able to rapidly and synchronously induce a site-specific DSB in Saccharomyces cerevisiae upstream of the PHO5 locus. This region of the genome is recognized for its chromatin organization, which is comprised of well-positioned nucleosomes. Utilizing MNase digestion of chromatin followed by paired-end fragment sequencing I was able to interrogate the order of chromatin changes that occur immediately following a DSB by generating a base-pair resolution map of the chromatin landscape. In wild-type cells, the first nucleosome left of the break was rapidly evicted. The eviction of this flanking nucleosome was dynamic and proceeded through an early intermediate chromatin structure where the nucleosome was repositioned in the adjacent linker DNA. Other nucleosomes bordering the break were also shifted away from the break; however, their loss was more gradual. These local changes preceded a broader loss of chromatin organization and nucleosome eviction that was marked by increased MNase sensitivity in the regions ~8 kb on each side of the break. While the broad loss of chromatin organization was dependent on the end-processing complex, Mre11-Rad50-Xrs2 (MRX), the early remodeling and repositioning of the nucleosome adjacent to the break was independent of the MRX and yKU70/80 complexes. I also examined the temporal dynamics of non-homologous end joining (NHEJ) mediated repair in a G1-arrested population. Concomitant with DSB repair, I observed the re-deposition and precise re-positioning of nucleosomes at the originally occupied positions. This re-establishment of the pre-lesion chromatin landscape suggests that a DNA replication-independent mechanism exists in G1 cells to preserve epigenome organization following DSB repair.

Mitotic homologous recombination (HR) is vital for accurate repair of DNA strand breaks caused by endogenous and exogenous sources. However, this high-fidelity repair pathway also can lead to genome rearrangements when dispersed sequences are used for repair. During normal growth, spontaneous DNA strand breaks are presumably generated during DNA replication and transcription, and from the attack by endogenous agents such as reactive oxygen species (ROS). Though the exact nature of endogenous lesions that initiate HR is not well understood, double-strand breaks (DSBs) rather than single-strand breaks (SSBs) are thought to be the main culprit. Because spontaneous HR events can lead to development of human diseases and sporadic cancers, identifying the primary type of DNA strand breaks, either DSBs or SSBs, is central to understanding how genome instability arises. Using the yeast Saccharomyces cerevisiae as a model system, the focus of this thesis is to delineate early molecular steps (DNA end resection and synthesis) during mitotic DSB-induced HR events and to perform comparative analysis of DSB-induced and spontaneous HR repair outcomes. To this end, the first part of this thesis examined the relative contribution of DNA end resection and DNA synthesis in determining the DSB-associated repair outcomes, such as distributions of crossover and noncrossover outcomes, as well as the length of a key recombination intermediate, heteroduplex (hetDNA). The main conclusion from this work is that both end resection and DNA synthesis are required to obtain normal DNA repair outcomes and hetDNA profiles. A unifying model is that decreased end resection reduces stability of strand invasion intermediates, limiting the extent of DNA synthesis and hence shortening hetDNA. The second and third parts of this work directly compared the molecular structures of HR outcomes associated with a defined DSB and with those arising spontaneously. Two different approaches were employed to systematically characterize the molecular structures of recombination intermediates in repair events. In the second part of this thesis, mapping of gene conversion events (nonreciprocal transfer of information that results from mismatch-repair activity) following allelic repair of a DSB revealed that DSB-induced HR events shared similar repair profiles with those of previously described spontaneous recombination events, confirming that DSBs are the main contributor to spontaneous HR. In the third part of this thesis, mapping of hetDNA in DSB-induced and spontaneous HR events in cells with normal and elevated ROS levels further confirmed that DSBs are the primary initiator of spontaneous HR. Mapping of hetDNA additionally revealed complexities within hetDNA associated with a defined DSB. Collectively, this work not only advances our knowledge of the fundamental molecular mechanisms of HR, but also provides in vivo experimental support for DSBs as the major physiological lesion that initiates spontaneous HR.

Topoisomerase 2 (Top2) is an enzyme that helps maintain genome integrity by resolving topological structures that arise during cellular processes such as replication and transcription. To resolve these structures, a Top2 dimer creates a transient double-strand break (DSB) in the DNA. Each subunit forms a phosphotyrosyl bond with the 5’ ends of the break, and this DNA-protein intermediate is called a Top2 cleavage complex (Top2cc). Following the passage of an intact duplex, Top2 re-ligates the DNA and is released to restore genome integrity. Top2cc stabilization by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. This thesis characterizes two novel top2 mutants, both of which are associated with a mutation signature characterized by de novo duplications. These duplication events are dependent on clean removal of the Top2cc from the DNA and DSB repair by nonhomologous end-joining. The first mutant (top2-FY,RG) was identified through a screen for etoposide hypersensitivity, and it generates a stabilized cleavage intermediate in vitro. The second mutant (top2-K720N) is the yeast equivalent of a somatic mutation in TOP2A identified in gastric cancers and choloangiocarcinomas that is also associated with a duplication mutation signature (ID17). Overall, the findings in this thesis are relevant for clinical use of chemotherapeutic drugs that target Top2 and have implications for genome evolution.

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.