Identification of novel tumor suppressors in the DNA damage response pathway and functional validation of them in vivo.
Maintenance of genomic integrity is of fundamental importance for the survival of all living organisms. This genomic integrity can be threatened during normal cell cycling as well as by external chromosomal insults, thus cell must sense the DNA damages and activate the DNA damage signal transduction pathways including cell cycle checkpoint control and DNA repair. The key players in DNA damage responses are the ATM(Ataxia telangiectasia-mutated), ATR(AT and rad3-related), Chk1 and Chk2 kinases in mammalian cells, which play an essential role in activating DNA damage responses via phosphorylation of many downstream targets following DNA damage. The final effectors are checkpoint controller(p53, Nbs1/Mre11/Rad50, Brcal) and DNA repair proteins(NBS1/Mre11/Rad50, Brcal). Significantly, many of genes involved in this pathway are implicated in human cancer prone diseases such as A-T(ATM-/-), Li-Farumeni Syndrome(LF; p53-/- or Chk2-/-); Familial Breast & Ovarian Cancer(Brca+/-), Nijimengen breakage syndrome(NBS1-/-), A-T like disorder(ATLD; Mre11-/-).
Since most of proteins mediate their functions through protein-protein interactions, the DNA damage response pathway is a web of protein-protein interactions. Therefore, these genes responsible for developing human cancer prone diseases will provide us to identify more proteins which mutations cause of sporadic cancer development. Here, in order to discover the novel tumor suppressors and to map the protein-protein interactions between known proteins. we developed and used a high-throughput yeast two-hybrid system("reverse proteomics"), starting with 15 proteins involved in the DNA damage responses. First, we mated the prey libraries(4.5×10^(7)) to 15 baits, subsequently the 980 surviving diploid cells in selection media have been identified. Initially, we cloned more than 20 novel genes out of 100 positive clones, which might be involved in DNA damage responses and now continue to analyze the remaining protein-protein interactions. In addition, more than 13 new novel protein-protein interactions were mapped in DNA damage response pathways. We will discuss the partial protein networks and function of novel proteins in DNA damage responses.