Rejoining of DNA double -strand breaks with complex end structures.

Abstract

Double-strand breaks (DSBs) are considered the most dangerous form of DNA damage because they are lethal to the cell if unrepaired, and can cause large-scale genome rearrangements if repaired incorrectly. Such rearrangements can lead to deregulation of tumor suppressor genes and oncogenes as observed in cancer. Restorative DSB repair mechanisms help to protect the genome from chromosomal rearrangements. Two DSB repair pathways, nonhomologous end joining (NHEJ) and homology-directed repair (HDR), are conserved throughout all kingdoms of life. HDR uses a homologous sequence as a template for repair. In NHEJ, the ends of the break are processed if necessary and ligated to restore the duplex. While NHEJ of DSBs with short compatible overhangs has been well studied, less is known about how breaks with long overhangs or damaged termini are rejoined. This dissertation focuses on NHEJ of DSBs with complex end structures using the yeast <italic>Saccharomyces cerevisiae</italic> as a model system. To create such breaks, we ligated annealed oligonucleotides onto the ends of a linearized plasmid, giving unprecedented control over the DSB ends. NHEJ was required when overhangs were short and annealing was energetically unfavorable. Longer overhangs could be rejoined efficiently in the absence of NHEJ, and their annealing was assisted by Rad52. Systematic modification of DSB termini determined which joint configurations required the NHEJ DNA polymerase Pol4 for gap filling. Pol4 was necessary when DSBs had unstable primer-template pairs at 3' overhangs, and the related mammalian Pol X family members could differentially complement <italic>pol4 </italic> mutation depending on the joint structure. The lyase domain of Pol4 was not required for removing 5' deoxyribosephosphate lesions from DSB termini and is likely redundant with NHEJ nucleases. Two genomic screening strategies that could identify such nucleases were therefore developed. Collectively, this work has shown that stabilizing the joint is a major function of NHEJ, and that the nature of the ends plays a critical role in determining which enzymes are involved in DSB rejoining. Paradoxically, this might help to maintain genome stability and prevent cancer through restorative repair of damaged termini, while enabling mutagenic joining of incompatible ends.