Palindrome fragility: a tool to understand the mechanism of break-induced replication involving sister chromatids
Homology-dependent repair of DNA double-strand breaks (DSBs) requires DNA synthesis that in different repair pathways ranges from several hundred base pairs to hundreds of kilobases. The long-range repair synthesis in eukaryotic cells was studied using experimental systems where only one homologous end of the broken molecule is available for the invasion. This process, coined break induced replication (BIR), is responsible for alternative lengthening of telomeres in cells lacking functional telomerase and for rescuing stalled and broken replication forks. In addition, BIR has a detrimental role in genome stability and contributes to the development of cancer and hereditary diseases by promoting loss of heterozygosity, genome rearrangements and mutagenesis. My laboratory made a crucial contribution towards understanding the mechanism of BIR involving recombination between disomic homologous chromosomes by characterizing the structure, mode of synthesis and genetic control of HO endonuclease-induced BIR. We identified a novel structure, a migrating replication bubble with asynchronous synthesis of leading and lagging strands that results in the accumulation of ssDNA. We determined that this mode of replication leads to a conservative inheritance of the new genetic material. We also found that Pif1and Srs2 are absolutely required for BIR synthesis. This was the first molecular characterization of the BIR structure that explained the unstable nature of BIR and its ability to promote mutagenesis, loss of heterozygosity, translocations, and copy number variations.
In this talk, I will present our recent results on identification and physical characterization of BIR initiated at the breakage-prone palindromic sequences. Using two-dimensional gel electrophoresis, we detect characteristic BIR structures that progress from both sides of the break formed at the location of palindromes and involve sister chromatids. This is another type of repair synthesis that differs from the HO DSB nuclease- or nickase-induced events and arguably represents a more natural situation in the cell. We demonstrate that palindrome-mediated BIR is initiated outside of the S-phase and can bypass chromosomal replication origins and centromere. Surprisingly, palindrome-mediated BIR is inaccurate in 30 kb window but becomes error-free at longer distances. I will describe the role of the key proteins that regulate sister-chromatid BIR and discuss implications of these yeast studies for the human genome stability.
Department of Biology
Department of Biology, Georgia Institute of Technology
Domain 1 - UMR 3348 - Genotoxic Stress and Cancer