Chromosomal analysis of DNA damage tolerance in mammalian cells using a novel approach

A major obstacle in the study of DNA damage tolerance is the lack of high-resolution methods for simultaneously assaying TLS and HDR during chromosomal replication. Traditional methods are based on monitoring the formation of mutations by DNA damaging agents in a reporter gene (e.g., HPRT), with the assumption that such mutations are caused by mutagenic TLS. However these methods are blind to accurate TLS, which is the main TLS pathway for some common types of DNA damage. Moreover, such methods are ineffective for measuring HDR. We, and others, have used plasmid-based model systems to study DNA damage tolerance via TLS and HDR. These model systems provided a wealth of insight into the mechanism of TLS and HDR, however, they do not obliterate the need to study TLS and HDR at the chromosomal level. To that end we are developing novel methods for the simultaneous analysis of TLS and HDR at a single nucleotide resolution in chromosomes of mammalian cells in culture. This is done using phage integrase, or a transposon, to promote the integration into mammalian chromosomes of plasmids carrying site-specific defined lesions. These systems are useful for the analysis of TLS and HDR in mammalian cells, and their involvement in pathological processes such as carcinogenesis.