p53 promotes maintenance of genetic information either by causing apoptosis of damaged cells, or by altering the cell cycle and repair pathways such that damage can be accurately repaired. The nematode Caenorhabditis elegans possesses only one p53 family member, CEP-1, that controls apoptosis and the cell cycle in response to genotoxic stress. Mutation in the meiotic gene him-5 increases nondisjunction of the X chromosome, resulting in increased frequencies of XO male and XXX Dpy progeny, and it affects the frequency of meiotic recombination on X. him-5 is allelic to the ORF D1086.4, which encodes a putative basic protein with no clear homologues or domain structure. The modest embryonic lethality (Emb) of him-5 mutants is dramatically increased by mutation of cep-1 but no change is seen in the proportion of XO male or XXX Dpy progeny. The synergistic effects of cep-1 and him-5 mutation are independent of CEP-1's DNA damage regulators and other meiotic mutants, and they do not involve deregulated apoptosis. cep-1; him-5 double mutants have abnormal chromatin morphology in diakinesis-arrested oocytes reminiscent of that seen in double strand break (DSB) repair mutants. This phenotype depends on the presence of SPO-11-induced meiotic DSBs, suggesting CEP-1 and HIM-5 function together to promote accurate recombination during meiosis. In support of this hypothesis, cep-1; him-5 show a significant reduction in crossover frequency between autosomal markers compared to wild-type or either single mutant alone, suggesting they function together to promote meiotic crossing over. The X chromosome nondisjunction in both him-5 and cep-1; him-5 is a result of failure of DSB formation and subsequent chiasma formation on the X. However, the embryonic lethality phenotype of him-5 and cep-1; him-5 is caused by a defect either downstream or in parallel to meiotic DSB formation. The diakinesis chromatin phenotype of cep-1; him-5 suggests this defect may be in meiotic DSB repair. This is confirmed by the fact that cep-1; him-5 animals show more persistent meiotic DSB-associated RAD-51 foci staining compared to wild-type, suggesting CEP-1 and HIM-5 may function in efficient resolution of SPO-11-induced DSBs during meiosis. A role for CEP-1 in promoting accurate repair of DSBs during meiosis may be related to p53's function in promoting faithful meiotic recombination in mammalian cells. HIM-5's role in DSB formation and repair suggests another mechanistic link between these recombination steps. Meiotic recombination is vital for genome stability, and characterization of the role of CEP-1 and HIM-5 will increase our understanding of the p53 family and genetic redundancy at multiple steps in this process.

Thesis (Ph. D.)--University of California, San Diego, 2002. / Vita. Includes bibliographical references (leaves 146-152).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (p. 114-125).

At the very center of sexual reproduction is meiosis. During meiosis, the formation of meiotic Double-Strand-Breaks (DBSs) and their repair by homologous recombination are widely conserved events occurring among most eukaryote species. Meiosis-specific DSB formation requires at least nine proteins (Spo11, Ski8, Rec102, Rec104, Mei4, Mer2, Rec114, Mre11/Rad50/Xrs2) in S. cerevisiae, and the resection of the DSB ends requires additional four proteins (Mre11/Rad50/Xrs2, and Sae2). Spo11 has been identified as the catalytic component of this DSB-initiating complex. However, the roles played by the majority of these proteins are not clear. I have purified the recombinant Spo11/Ski8/Rec102/Rec104 complex, characterized its DNA binding ability as well as its cleavage activity on supercoiled plasmid DNA. Sae2 functions in both meiotic and mitotic repair of DNA double-strand breaks (DSBs) in S. cerevisiae. In vivo experiments have shown that Sae2 collaborates with the Mre11/Rad50/Xrs2 (MRX) complex in DNA end processing. Our laboratory previously showed that recombinant Sae2 exhibits endonuclease activity on single-stranded DNA and single-strand/double-strand DNA junctions using purified proteins in vitro. The MRX complex stimulates Sae2 endonuclease activity on single-stranded DNA close to single-strand/double-strand junctions, through its endonucleolytic activity. However, Sae2 contains no conserved typical nuclease domain, and it only shares very limited homology with its human functional counterpart CtIP. To characterize Sae2 and the active sites responsible for its nuclease activity, I used partial proteolysis and site-directed mutagenesis to analyze the protein. Biochemical assays in vitro show that acidic residues in the central domain play an important role in Sae2 endonuclease activity. Sae2 has also been shown to be phosphorylated by CDK (Cyclin-Dependent Kinase) during the S and G2 phases of the cell cycle, as well as by Tel1/Mec1 upon DNA damage. These modifications are essential for the function of Sae2 in DNA repair, but the function of these modifications are not clear. I have demonstrated that, in the presence of MRX, Sae2 (5D/S267E) mimicking constitutive phosphorylation by CDK and Mec1/Tel1 can assist the 5’ to 3’ exonuclease Exo1 significantly in 5’ end resection by suppressing the inhibitory effect of Ku. These results suggest that Sae2 is a critical switching protein which determines the choice between HR and NHEJ in yeast cells upon DNA damage. / text

The present study combines modern jet reconstruction algorithms and particle identification (PID) techniques in order to study the enhancement of proton/pion ratio at mid transverse momentum ([mathematical symbols] 1.5 - 4.0 GeV/c) observed in central Au + Au collisions at [mathematical symbols] = 200 GeV. The ratio enhancement is thought to be caused by recombination processes and/or parton fragmentation modification of jets in relativistic heavy ion collisions. The fragmentation modification hypothesis is tested in this analysis by reconstructing and selecting energetic jets presumably biased to fragment outside of the medium created in Au + Au collisions and comparing their particle composition to the recoiling (medium-traversing) jets. The bias assumption is confirmed by comparing jets in central collisions, where the effect of proton/pion enhancement is present, with peripheral ones where no medium effects are expected. The selected jets are reconstructed by using the anti-k[subscript T] algorithm from the modern FASTJET package. The PID in the p[subscript T] region of interest is possible by combining measurements of the particles' energy deposition and velocity from the Time Projection Chamber and the recently installed (2009-2010) Time of Flight detectors at STAR. The acceptance of these detectors, [eta] < 1.0 and full azimuth, make them extraordinary tools for correlation studies. These features allow for the measurement of relative azimuth ([phi] [subscript jet] - [phi] [subscript pion,proton]) distributions by using the selected jet axis in order to disentangle the uncorrelated background present in the high multiplicity heavy ion collisions. The proton/pion ratios in two different centrality bins and p[subscript T] = 1.2 - 3.0 GeV/c are presented for biased (vacuum fragmenting) jets and their recoiling counterparts / text

published_or_final_version / abstract / toc / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

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