Mechanism and Regulation of DNA Recombination

Baylor College Of Medicine, Houston TX

Investigators

Abstract

Recombination is essential for repairing DNA double-strand breaks (DSBs) and maintaining genomic stability. DSBs can be repaired through non-homologous end-joining (NHEJ) or homologous recombination (HR). Even minor deficiencies in DNA recombination can result in cancer, immune deficiencies, or other severe diseases. The goal of the proposed research is to understand the mechanisms and regulation of DNA recombination using the model organisms of budding and fission yeast. We will focus on three areas of research: 1. The initial processing of DNA double-strand breaks (DSBs) into single strands, a process termed 'DNA end resection', is the critical first step in homologous recombination. It is necessary for the loading of damage response and repair proteins. Systematic studies of resection have thus far been conducted only in euchromatin. We propose to study this process in constitutive heterochromatin, which constitutes a large fraction of eukaryotic genomes. We have designed multiple assays to study DSB ends resection and repair within heterochromatin using a fission yeast assay. 2. Repair of one-ended double-strand breaks (DSBs) can initiate extensive repair-specific DNA synthesis. This pathway, known as break-induced replication (BIR), is facilitated by Polδ/Pif1 helicase-driven D- loop migration. BIR is crucial for the maintenance of telomeres in telomerase-negative cancers, which constitute approximately 5-10% of all cancers. It is also implicated in many genomic rearrangements and in the mitotic DNA synthesis occurring at under-replicated genomic loci. Two fundamental questions about BIR will be addressed in this application: How does lagging strand synthesis proceed during BIR? Second, what mechanisms restrict efficient BIR to subtelomeric regions? Understanding the constraints that limit BIR to areas near telomeres is vital, as BIR is a highly mutagenic pathway prone to template switching. 3. Repair of DSBs can be associated with templated insertions, a type of genome rearrangement where a DNA segment is inserted into a DSB. These copy number variations (CNVs) are among the most common in cancer genomes, yet their formation mechanism remains unclear. Templated insertions can occur at programmed breaks at VDJ locus or at HO or CRISPR/Cas9 endonuclease-induced breaks and typically range from approximately 50 bp to 1 kb in size. Templated insertions are mediated by Ku-mediated non-homologous end joining (NHEJ). We have developed a simple and cost-effective amplicon sequencing-based assay, termed 'Break-Ins', which allows systematic studies of templated insertions. Using this assay, we screened for mutants with a high level of templated insertions. We propose to study in-depth a group of identified mutants that are closely related to human neurodegenerative diseases and cancer. The primary goal is to understand the cellular conditions that drive the formation of templated insertions. Additionally, using 'Break-Ins', we identified mutants and conditions that lead to elevated release of mitochondrial DNA (mtDNA) and its transfer to the nucleus. Our goal is to understand the mechanisms that restrain the release of mtDNA from mitochondria and its transfer to the nucleus.