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Loss of DNA damage-induced G2 checkpoint control is associated with genome instability, tumour formation and the therapeutic response of tumours to genotoxic agents. The large 3418 residue protein encoded by BRCA2 – heterozygous germline mutations in which predispose to cancer - has recently been implicated in G2 checkpoint maintenance. However, the mechanistic basis of BRCA2’s role in the G2 checkpoint remains unknown. The overall aim of my research is to understand the mechanism by which BRCA2 regulates the G2 checkpoint. Domain mapping studies, using overlapping fragments encoding the full-length BRCA2 protein, carried out in our laboratory suggest that BRCA2’s function at the G2 checkpoint is mediated through regions that span BRCA2 amino acids (aa) 1-454 and aa 2438-2824. My research has focused on understanding how these two regions contribute to G2 checkpoint function through the interrogation of two novel interactions of BRCA2 mediated at these regions. My experiments have identified that BRCA2 interacts with the serine/threonine kinase ATR (aa 2438-2824) and the deSUMOylase SENP1 (aa 1-454). ATR is known to play a key role at the G2 checkpoint and my results identify that loss of BRCA2 leads to a reduction in ATR activity at sites of damage. This leads to a downstream attenuation in the phosphorylation of Chk1 – an important effector protein of the G2 checkpoint. BRCA2 is known to function in DNA repair and is recruited to sites of DNA damage, where it displaces RPA bound to exposed single-strand DNA. RPA is required to localise and activate ATR during the G2 checkpoint and therefore I hypothesise that BRCA2’s role at the checkpoint is to substitute for RPA and mediate ATR activation. I have also shown that SENP1 interacts with BRCA2 at a region within the N-terminus (aa 290-454). SUMOylation has been increasingly recognised as an important post-translational modification (PTM) in the context of the DNA damage response, and SENP1 is involved in reversing this PTM. I have shown that BRCA2 is SUMOylated. Moreover, I have shown that loss of SENP1 prevents BRCA2 from being recruited to chromatin in a timely manner after DNA damage and that SENP1 depletion exacerbates the loss of G2 checkpoint maintenance seen in BRCA2 deficiency. Taken together, this work reveals novel insights into the mechanism by which BRCA2 maintains genomic stability through enforcement of the G2 checkpoint. This new knowledge has the potential to translate into a better understanding of how mutations in BRCA2 may lead to cancer.

<p>The plant cyclin-dependent kinases (CDKs) are subjected to the regulation by the interactors/inhibitors of CDK (ICKs). Seven members of the ICK family are known in the plant <i>Arabidopsis thaliana</i>. It has been shown that the N-terminal region of ICK1 makes it unstable in plants, although the mechanism was unknown.</p> <p>In this thesis, to determine role of the N-terminal region in other ICKs, full length ICK2 to ICK7 were compared to truncated mutants lacking the N-terminal region. Results from yeast two-hybrid studies suggest that not all the N-terminal regions in different ICKs have a role similar to the N-terminal region of ICK1. Studies of a set of HA-tagged ICK1 deletion mutants in yeast mapped the critical sequence for ICK1 degradation to the N-terminal 21^st - 40^th amino acid residues. Overexpression of deletion mutants in Arabidopsis also showed that deletion of the 20-amino-acid region of ICK1 lead to a high level of HA-tagged mutant protein, supporting that this region plays a major role in ICK1 degradation <i>in vivo</i>.</p> <p>Treating yeast cells expressing HA-tagged ICK1 with the 26S proteasome inhibitor MG132 moderately increased the level of ICK1 protein, suggesting that 26S proteasome may be involved in the degradation of ICK1. To determine the possible involvement of the two E3 complexes, the Skp1-Cullin-F-box (SCF) complex and anaphase promoting complex/cyclosome (APC/C), a set of yeast mutants defective in either SCF complex or APC/C, were used to express ICK1, ICK1^ÎN20 (ICK1 lacking the N-terminal 20 amino acids) and ICK1^ÎN40. ICK1^ÎN40 showed a very high level of expression in SCF defective mutants, but not in APC/C defective mutants. However, ICK1 and ICK1^ÎN20 did not accumulate to a high level in either of the two types of mutants. These results suggest that two pathways are involved in the degradation of ICK1.</p> <p>Results from this study provide new understanding regarding the role of N-terminal region of ICK1 in conferring protein instability, and the differences among ICKs. They also raise new questions for future investigation on this family of plant cell cycle regulators.</p>

The decision to initiate division is very important, as once cells have initiated division they are committed to complete it. In Saccharomyces cerevisiae, commitment to a new round of cell division occurs at a regulatory point in late G1 called START. Progression through START requires the activation of the cyclin dependent kinase Cdc28p by the G1 cyclins. G1 cyclins in complex with Cdc28p activate the transcription of approximately 100 genes involved in the G1 to S transition and degradation of Sic1p, an inhibitor of B type cyclins, and thus are important for initiation of DNA replication. Despite the widely studied role of regulatory cyclins and cyclin dependent kinase in the G1 to S transition, how cells determine when to initiate DNA replication is poorly understood. We have identified several gene products, which when overexpressed, cause cells to initiate DNA replication faster than wild type. Here we discuss the role of DCR2 (Dosage dependent Cell cycle Regulator), GID8 (Glucose Induced Degradation) and KEM1 (Kar-Enhancing Mutation) in the regulation of START. Over expression of DCR2 and GID8 accelerates initiation of DNA replication. Cells lacking both these genes delay initiation of DNA replication. Genetic analysis suggests that Gid8p functions upstream of Dcr2p to promote START. Further, we show that DCR2, which codes for a metallo-phosphoesterase, might regulate completion of START by affecting degradation of Sic1p. Over expression of DCR2 lowers the half-life of Sic1p without altering the expression of Cln2p. The evidence suggests that Dcr2p affects START completion through dephosphorylation of Sic1p. KEM1 is a Saccharomyces cerevisiae gene, conserved in all eukaryotes, which codes for a 5’-3’ cytoplasmic exonuclease. This exonuclease is involved in exiting mitosis, by degrading the mRNA of the mitotic cyclin CLB2. Besides its role in mitotic exit, an enzymatically inactive version of Kem1p can accelerate the G1 to S transition and initiation of DNA replication when over expressed. This result suggests that Kem1p might have a previously unrecognized role in the G1 to S transition independent of its exonuclease activity, and supports the notion that Kem1p is a multifunctional protein with distinct and separable roles.

Includes bibliographical references (p. 118-134).

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

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published_or_final_version / abstract / toc / Molecular Biology / Master / Master of Philosophy

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