During female meiotic prophase in many organisms, a specialized chromatin structure is formed in the oocyte nucleus. This structure is known as the karyosome, and has been proposed to be important for the formation of the female meiotic bipolar spindle. However, how the karyosome is formed and maintained is not very well understood. To identify proteins involved in the formation and maintenance of the karyosome, I carried out a cytological screen on a collection of 220 mutant fly lines for mutants that were defective in karyosome morphology. The screen identified 46 mutants on the X and 2nd chromosome with abnormal karyosomes. Genetic analysis of these 46 mutants, followed by molecular analysis of one mutant, identified SRPK (SR Protein Kinase) as a protein that is important for the proper formation of the karyosome. NHK-1 (Nucleosomal Histone Kinase 1) was previously identified as a protein that is essential for the formation of the karyosome via its phosphorylation of BAF (Barrier-to-Autointegration Factor). NHK-1 phosphorylation of BAF leads to the release of chromatin from the nuclear membrane, an essential step for the formation of the karyosome, however, the regulation of this process is unclear. In order to identify genes that interact with NHK-1, I carried out a genetic modifier screen using a semi-lethal allele of NHK-1, NHK-1trip. After screening a collection of 44 deficiencies located on the 2nd chromosome, I identified a genetic region (44B8-44D1) containing a gene that interacts with NHK-1 and, when gene dosage is halved, enhanced the semi-lethal phenotype of NHK-1trip.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13537号 / 論医博第2277号 / 新制||医||1065(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 近藤 玄, 教授 齋藤 潤 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Abstract Genetic information is stored in the base sequence of DNA. As DNA is often damaged by radiation or reactive chemicals, cells have developed mechanisms to correct the DNA lesions. These mechanisms involve recognition of damage, DNA repair and cell cycle delay until DNA is restored. Failures in the proper processing of DNA lesions may lead to mutations, premature aging, or diseases such as cancer. In this thesis study the human topoisomerase IIβ binding protein 1 (TopBP1) was identified as the homolog of budding yeast Dpb11 and fission yeast Cut5. TopBP1 was found to be necessary for DNA replication and to associate with replicative DNA polymerase ε. TopBP1 localised to the sites of DNA damage and stalled replication forks, which suggests a role in the DNA damage response. TopBP1 interacted with the checkpoint protein Rad9, which is a part of a protein complex whose function includes tethering proteins to sites of DNA damage. This supports a role for TopBP1 in the early steps of checkpoint activation after DNA damage. TopBP1 also interacted with the tumour suppressor protein p53 in a phosphorylation dependent manner. In addition, the data support a role for TopBP1 outside of S-phase. During M-phase, TopBP1 was found to localise to centrosomes along with the tumour suppressor proteins Brca1 and p53. Analysis of the expression of TopBP1 in mouse tissues suggested that TopBP1 may also play a role during meiosis. The localisation pattern of TopBP1 in mouse meiotic spermatocytes resembled that of many proteins functioning during meiotic recombination. For example, co-localisation of ATR kinase and TopBP1 was observed during meiotic prophase I. In accordance with the findings from mouse studies, the analysis of a cut5 mutant during yeast meiosis showed that Cut5 is essential for the meiotic checkpoint. These results strongly suggest that TopBP1 operates in replication and has checkpoint functions during both the mitotic and meiotic cell cycles.