No description available.

No description available.

No description available.

No description available.

In eukaryotes, chromosomes are non-randomly positioned within the nucleus.  The perinuclear localization of <i>S. cerevisiae </i>telomeres provides a useful model for studying mechanisms that control chromosome positioning.  In budding yeast, telomeres tend to be localized at the nuclear periphery during early interphase, but following S phase they delocalize and remain randomly positioned within the nucleus.  In this thesis, I investigate whether DNA replication causes telomere dislodgment from the nuclear periphery. First, using live-cell fluorescence microscopy I show that delaying DNA replication causes a corresponding delay in the dislodgement of telomeres from the nuclear envelope, demonstrating that replication of individual telomeres causes their delocalization.  Second, I show that telomere dislodgment is not simply the result of recruitment of telomeres to a replication factory that is formed in the nuclear interior, since I found that telomeric DNA replication can occur either at the nuclear periphery or in the nuclear interior.  The telomere binding complex Ku is one of the factors that establishes telomere localization to the nuclear envelope.  Using a gene locus tethering assay,  I show that the Ku-mediated telomere localization pathway is inactivated after DNA replication. Based on these findings, I propose that DNA replication causes telomere delocalization by triggering stable repression of the Ku-mediated anchoring pathway.  In addition to maintaining genetic information, DNA replication may therefore regulate subnuclear organization of chromatin.

This paper revisits Grunfeld's well-known investment data, one of the most widely used data sets in all of econometrics, on the occasion of their 50th anniversary. It presents, apparently for the first time after the publication of the original Chicago Ph.D. thesis, the full data set and points out errors and inconsistencies in several currently available versions. It also revisits a number of empirical studies from the literature of the last five decades. / Series: Research Report Series / Department of Statistics and Mathematics

DNA replication is a key process required by organisms during cell division, with a concomitant requirement for genome synthesis by DNA polymerases. Biotechnological exploitation of thermostable DNA polymerases for DNA amplification by the Polymerase Chain Reaction (PCR), provides a significant market for novel enzymes or those with improved properties. An approach was taken to isolate alternative thermostable DNA polymerases, by enriching thermophilic bacteria from a novel thermal environment, aerobically spoiling silage. In addition, a novel DNA polymerase (Abr polBl) was cloned from the thermoacidophilic archaeon, Acidianus brierleyi, with the intention of characterising its in vivo role and application to PCR. Protein sequence analysis suggested a proofreading (high fidelity) DNA synthesis activity most related to polBl DNA polymerases from Crenarchaeota. Abr polBl was heterologously expressed in bacteria and protein purified to homogeneity. Biochemical assays confirmed high-temperature DNA polymerase and 3'-5'exonuclease activities of Abr polBl, with an accompanying proofreading ability. Sequence analysis, processivity, strand displacement and lesion bypass activities indicated potential roles in genome replication and DNA repair. Abr polBl could not amplify DNA under a range of PCR conditions, presumably following its low intrinsic thermostability. Biophysical analyses confirmed irreversible unfolding of Abr polBl at temperatures required for PCR. Supplementation with organic compounds and ionic salts stabilised Abr polBl, promoting retention of conformational stability and DNA synthesis activity following thermal incubation, but could not promote DNA amplification with Abr polB 1.

<p>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.</p><p>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. </p><p>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. </p><p>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.</p><p>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.</p> / Dissertation

In an effort to achieve replicative immortality, human cancer cells must avoid the constant telomere attrition that accompanies DNA replication. Cancer cells accomplish this by employing mechanisms to lengthen their telomeres. Approximately 10 percent of all cancers utilize the Alternative Lengthening of Telomeres (ALT) pathway to maintain telomere length. Although ALT is known to rely on homologous recombination between two telomeric sequences, the exact mechanism and regulators of the ALT pathway remain elusive. As common fragile sites, telomeres pose a challenge to the replication machinery. This replication challenge is exacerbated in ALT cells due to defects in nucleosome assembly, suggesting the importance of managing replication stress at telomeres in these cells. ATR (ataxia telangiectasia and Rad3-related) is an important kinase in the response to replication stress. The work in this thesis demonstrates that ATR is also a key mediator of ALT activity. Due to the highly recombinogenic state of ALT telomeres, these cells depend on ATR activity. In fact, we illustrate that small molecule inhibition and siRNA mediated loss of ATR disrupts ALT activity and promotes cell death specifically in ALT positive cancer cells. Although we establish ATR as a critical regulator and effective therapeutic target in ALT cancers, the exact mechanism of ATR in this pathway remains elusive. Recently, the chromatin remodeling enzyme SMARCAL1 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin subfamily A-like protein 1) was identified as one of the most abundant proteins bound to sites of replication stress. We demonstrate by combined immunofluorescence-FISH and chromatin immunoprecipitation that SMARCAL1 associates with ALT telomeres to resolve replication stress and maintain telomere stability. Specifically, we illustrate that siRNA mediated loss of SMARCAL1 in ALT cancer cells results in persistently stalled replication forks that collapse into DNA double strand breaks, which promotes the formation of chromosome fusions. Ultimately, we illustrate that loss of SMARCAL1 in ALT cancer cells promotes genomic instability through telomere dysfunction. Although great strides have been made in defining the ALT mechanism, the drivers of this pathway remain elusive. These studies highlight the importance of replication stress in both activation and maintenance of the ALT pathway. Our data demonstrate chronic replication stress as a key feature at ALT telomeres. Importantly, we were able to exploit this feature to identify a novel therapeutic avenue for ALT positive cancers.

The cell cycle is a highly orchestrated developmental process that eventually leads to the reproduction of a cell. In metazoans, it is driven by the successive activation of cyclin-dependent kinases (Cdk) and proper coordination of cell cycle transitions and processes ensure genomic stability. DNA replication takes place during S phase to faithfully duplicate a cell’s genetic material. In eukaryotes, S phase onset involves the initiation of numerous licensed replication origins across the genome and requires the activities of two protein kinases, S phase-Cdk and Cdc7. In this thesis, I present work relating to the role of the S phase-promoting kinases in DNA replication and S phase regulation. Using the cell-free system of Xenopus egg extracts, a small molecule inhibitor of Cdc7, PHA-767491, was characterised. PHA-767491 was then used to demonstrate that Cdc7 executes its activity early in S phase before the Cdk-dependent step. Cdc7 is not rate limiting for the progression of the replication timing programme once its essential function has been executed, unlike S-Cdk whose activity is required throughout S phase. Protein Phosphatase 1 (PP1) was identified as a modulator of Cdc7 activity in egg extracts, which rapidly reverses Cdc7-dependent phosphorylation of chromatin-bound Mcm4 and likely functionally lowers Cdc7 activity during an etoposide-induced checkpoint response. This provides a novel mechanism for regulating Cdc7 by counteracting its activity on essential replication substrates in the event of replicative stress. In the second part of the thesis, the design strategy for generating a Cdc7-conditional knockout mouse (cko) is outlined and results from the screen for a transgenic founder are presented. A Cdc7-cko mouse will be a valuable tool to further dissect Cdc7 function and regulation in mammalian cells. In the final section, S phase entry and progression in mouse embryonic fibroblasts lacking both Cdk1 and Cdk2 was examined. Contrary to expectations, Cdk1/Cdk2 double knockout cells can enter S phase in the absence of detectable S phase-Cdk activity. S phase progression, however, was inefficient. Cdc6 and cyclin E1 proteins were found to accumulate in high levels in these cells. The exact function(s) and mechanism(s) for these observations remain to be discovered. With this work, I hope to provide additional insight into the roles and regulation of S phase kinases in eukaryotic DNA replication.