DNA Damage Responses of Mitotic Cells Following Laser Micro-irradiation.

摘要

The natural occurrence of DNA damage has resulted in the evolution of processes that maintain cellular genomic integrity. These include (a) recognition of damaged DNA, (b) cell cycle control that halts progression through mitosis and thus block passage of compromised DNA to the next generation of cells, and (c) various mechanisms of DNA damage repair. Defects associated with DNA repair are associated with developmental disorders, cancer and accelerated aging. Double strand breaks (DSBs) are amongst the most serious forms of DNA lesions. A single double strand break can lead to cell death if left unrepaired. Further, double strand breaks can lead to chromosomal translocations that are highly associated with tumorigenesis. Thus, interphase DSB responses are the focus of several investigations. Few studies, however, have investigated the DSB responses of cells in mitosis.;My studies have focused on the use of laser microsurgery to induce DSBs on mitotic chromosomes, and to examine the subsequent DNA damage response mechanisms. These studies have led to the determination of laser parameters that result in consistent double strand break and pyrimidine dimer production as well as characterization of the change in chromatin ultrastructure following laser irradiation. In addition, a relationship between mitotic DNA damage and retention of the mitotic marker, phosho-histone H3 Serine 10, has been identified. My results show that contrary to other studies, proteins downstream of DSB recognition (Ubiquitin, BRCA1 and 53BP1) accumulate at laser-induced mitotic DNA damage sites. Furthermore, both homologous recombination and non-homologous end joining (NHEJ) repair pathways are activated. However, downstream activation is only observed for NHEJ. Additionally, cells deficient in DNA-PKcs have demonstrated the ability to repair via EdU incorporation, thus, suggesting the activity of alternative-NHEJ. In addition, activation of the nucleotide excision repair, single strand break repair, and Fanconi Anemia pathways have been detected in mitotic cells. The results also demonstrate that DNA damage in mitosis is repaired less effectively than DNA damage induced in G1. For the purposes of this thesis, "effective repair" means that a cell can undergo a subsequent cell division after it has repaired the damage produced in the previous mitosis.