| Summary: | Bloom's syndrome (BS) is a rare autosomal recessively transmitted disorder. The gene mutated in BS encodes the DNA helicase, BLM which is a member of the RecQ family. BLM is thought to have a role in the processing and reinitiation. of stalled replication forks. BLM is able to unwind a large array of DNA substrates of different structures formed at stalled replication forks but has a ' preference for unwinding Holliday junctions (HJ). BLM is considered to have a' role in DNA repair and the DNA damage response as it is a downstream target of sensor proteins ATM and ATR. The initial objective of-this study was to . investigate the role of Xenopus BLM in egg extracts during early Xenopus cell cycles Results showed that XBLM protein levels increase following aphidicolin induced replication arrest and that XBLM is modified by phosphorylation by PIKK kinases. I also looked at XBLM protein levels during early stages of Xenopus development and find that XBLM protein exists as a modified form in the matured egg and that it disappears at mid-blastula transition during early stages of Xenopus development. Due to the publication of a paper that reported results of the same experiments that I had hoped to later work on, I began to work on another protein that is thought to be cell cycle regulated, XNP95. Mouse nuclear phosphoprotein 95kDa (NP95~ was first identified as a cell cycle regulated protein whose regulated expression is abolished in thymic lymphoma cells. NP95 is a novel nuclear protein with an open reading frame consisting of 782 amino acids, which contains a ubiquitin binding domain, a leucine zipper motif, a zinc finger motif, a potential ATP/GTP binding site, a putative cyclin A1E-Cdk2 phosphorylation site, retinoblastoma protein (pRb) binding motifs, and a RING finger domain as well as an SRAlYDG domain. In this thesis I attempted to characterise the role'of XNP95 protein in early Xenopus cell cycles. The results in this study showed that XNP95 protein is not modified during the early stages of Xenopus development, and that it is localised to the nucleus of the oocyte. \fI.{I1en examining XN~~5 protein in Xenopus LSS extracts I find that XNP95 is a chromatin bound protein and that chromatin binding is cell cycle regulated, peaking at S phase and then disappearing upon onset of mitosis. Results also suggest that the increased association of XNP95 on chromatin seems to occur after the assembly of the pre-Reo I also investigated potential XNP95 modification and find that XNP95 seems capable of autoubiquitination although I find no evidence to suggest that it can be phosphorylated. Finally, results show that immunodepletion of XNP95 inhibits replication and that replication is not restored with recombinant XNP95 protein. The results of this thesis therefore suggest a potential role of XNP95 in the regulation of DNA replication.
|
|---|
