DNA damage response is vital to genome maintenance, cell survival and successful transmission of genetic information to daughter cells. This response is extremely important since DNA is subject to damage daily either by endogenous metabolic errors and byproducts or by exposure to genotoxic agents. Different types of lesions are formed as a result of such insults to the DNA; the most toxic of such lesions are those that affect both strands of the double-helix.
During my dissertation work, I studied cellular response to DNA lesions such as double-strand breaks and interstrand crosslinks using the model system Drosophila melanogaster. Double-strand breaks are repaired primarily by two mechanisms: homology mediated repair (HR) and nonhomologous end joining (NHEJ). Here I discuss the importance of homology mediated repair by studying repair defects in mutants defective for either of the two genes: (1) nbs gene encodes for the protein Nibrin, which is part of a well characterized protein complex MRN, comprising two other proteins Mre11 and Rad50 (2) okra encodes the Drosophila homolog of the Rad54 protein. While the MRN complex is hypothesized to be required during early steps of HR such as break resection, Rad54 is believed to be involved in chromatin remodeling and facilitating the role of the strand invasion protein, Rad51. I have addressed several questions here about the function of MRN in responding to double-strand breaks, using mutations in the nbs gene. Since the NBS protein is known to target the MRN complex to the nucleus, study of NBS in isolation should be reflective of the nuclear function of the MRN complex. The requirement of MRN for NHEJ and/or HR appears to differ in different organism. I found that Drosophila NBS is required for HR and not NHEJ. In addition, I found that in contrast to other studies, MRN may function in late steps of HR, post break resection in Drosophila. Study of defects in responding to DNA damage, specifically double-strand breaks (DSBs), in haploinsufficient nbs mutant backgrounds provided valuable clues into underlying molecular mechanisms that lead to carcinogenesis in human carriers of nbs mutation.
I tested to verify if DmRad54 is functionally conserved. This study showed that not only does DmRad54 facilitate DmRad51 function during first round of strand invasion, but it is also required multiple times while repairing the break, during the several rounds of strand invasion and synthesis that is characteristic of HR in pre-meiotic germline cells in Drosophila.
The second type of toxic lesion discussed here are the interstrand crosslinks (ICLs). Multiple repair mechanisms integrate to repair interstrand crosslinks in the bacteria Escherichia coli and the budding yeast Saccharomyces cerevisiae. Nucleotide excision repair (NER) and HR proteins are required for ICL repair, among others. Also, since DSB intermediates are formed while resolving ICLs, HR proteins seem to be integral in responding to crosslinks. I tested mutants defective in two genes, mus301 and mus302, both of which are hypersensitive to crosslinking agents, for defects in DSB repair. I found that while mus302 mutants, which have previously been implicated in NER, can repair double-strand breaks normally; mus301 mutants are severely defective in HR, when the only available homologous template for repair is the sister chromatid.