PARENT SESSION
1:30 PM to 3:30 PM
Monday, April 22, 2002
Poster Session 15 DNA Damage and Repair II
Room: Nevada Exhibition Center

(P20-200) The role of novel polyubiquitin chains in DNA post-replication repair and mutagenesis in mammalian cells .

Brun, Jan^*,1, Chiu, Roland², Gray, Douglas¹, Wouters, Brad², ¹ Department of Biochemistry: Human and Molecular Genetics, Ottawa, Canada² Department of Radiation Oncology, Maastricht, Netherlands

ABSTRACT-
Ubiquitin is a pleiotropic molecule involved in a variety of biological processes such as cell cycle control, endocytosis, proteolysis and DNA repair. Ubiquitin has 7 lysine residues, many of which can be used in the formation polyubiquitin chains. In particular, polyubiquitin chains linked through lysine 63 play a role in signal transduction during DNA repair processes. In yeast, this role is exemplified by the post-replication repair (PRR) pathway in which the K63 linked polymers mediate the lesion bypass of UV and MMS induced DNA damage. PRR is required when cells with damaged DNA escape the G1 checkpoint and polymerase stalls upon arriving at the site of a lesion. At this point alternative DNA repair polymerases are recruited for bypass of the DNA lesion. Bypass of these lesions may be error-free or error-prone, but in general the alternative polymerases have a much lower fidelity than polymerase . We have examined the role of the K63 linked ubiquitin polymers in mammalian cells by generating A549 cells stably expressing a series of ubiquitin mutants. The expression of a UbK63R transgene resulted in overt sensitization of these cell lines to cisplatin but not to UV or x-ray induced DNA damage. The G1 checkpoint may partially protect A549 cells (which have wild type p53) expressing UbK63R against mutation and death by allowing time for repair of lesions before entry into S-phase. This would prevent the recruitment of low fidelity translesion polymerases. To test this possibility, we plan to express the UbK63R transgene in isogenic human cell lines proficient or deficient in p21. We hypothesize that the p21 deficient cells, which are unable to block in G1, will require significantly more lesion bypass than the isogenic parental cell line. Finally, in order to study the role of this pathway in carcinogenesis, the mutant A549 cell lines were assayed for mutation frequency at the Hprt locus following treatment with DNA damaging agents. Cells expressing the K63R mutation showed increased mutagenesis and as a result we have generated transgenic mice expressing the K63R mutant form of ubiquitin to more directly test its role in cancer development.

KEYWORDS: ubiquitin, UV, p21, mutagenesis