The goals of the proposed research are to clarify the mechanisms and genetic consequences of mismatch repair double-strand break (DSB)- induced gene conversion, UV damage induction, and delayed genomic instability induced by low-dose ionizing Radiation, and to understand the effects of transcription on DNA damage induction, mismatch repair, and recombination. DNA repair and genetic recombination are ubiquitous and fundamental cellular process that are involved, for example, in gene regulation, development, and in the genetic changes associated with cellular transformation and tumor progression. Recombination is simulated by many agents that damage DNA including UV light, ionizing radiation, and chemicals; these agents are also known to be mutagenic and carcinogenic. Both recombination and DNA repair are influenced by transcription, and mismatch repair defects are common in many forms of cancer.
Aim1 focuses on the repair of loop and single-base mismatches in extrachromosomal recombination intermediates in mammalian cells. We will investigate factors involved in loop-specific mismatch recognition, such as loop length and structure, loop repair tract lengths, and the influence of transcription on single-base and loop repair efficiency and bias.
Aim 2 is designed to determine how factors such as allele proximity, relative positions of recipient and donor alleles, and transcriptional activity influence donor allele choice during spontaneous, DSB-induced, and UV -induced gene conversion in direct repeats in mouse chromosomal DNA. DSBs will be induced in chromosomal DNA using the highly specific I-SceI nuclease system.
Aim 2 is also designed to clarify whether these factors influence the relative rates of conversion and other modes of homologous recombination, such as deletion between direct repeats. Currently there is no information about donor preference in mammalian cells; the proposed experiments will provide a first view of this important aspect of conversion, which has relevance to recombination mechanisms and to the stability of mammalian genomes. Early reports indicated that initial UV damage levels were similar in transcribed and nontranscribed strands, but we recently found significantly higher damage levels in transcribed strands of highly active genes than in nontranscribed strands. The goal of Aim 3 is to determine whether differential damage in transcribed and nontranscribed strands is UV dose- and/or transcription level-dependent.
Aim 4 focuses on the induction of delayed genomic instability by ionizing radiation. WE will determine whether cells displaying delayed instability exhibit altered mismatch repair, mutagenesis, or recombination phenotypes. Nonselective assays will be employed to allow temporal analysis of delayed instability phenotypes as a model for tumor progression. Together the proposed projects will provide insight into the relationships among DNA damage and repair, recombination , mismatch repair and transcriptional effects on these processes, as well as improving our understanding of delayed genomic instability induced by ionizing radiation.
