Causes and consequences of genome instability and chromosome rearrangements

About This Grant

Abstract: This R35 MIRA application represents the five-year program plan for my laboratory. The overarching goal of my laboratory research program focuses on how DNA double-strand breaks (DSBs), a particularly toxic form of DNA damage, induce genome instability and chromosome rearrangements. DSBs are predominantly repaired by non-homologous end joining or homologous recombination. Both pathways are critical for embryonic development, and loss of either increases chromosome rearrangement formation and cancer predisposition. Defects in DSB repair are also potent therapeutic targets, either by traditional chemotherapeutics overwhelming cells with damage or more targeted drugs developed to target cells with specific pathway mutations. In previously published work from my laboratory supported by my NIGMS R01GM134537, we dissected the contribution to R loop metabolism on DSB repair and aberrant CR formation in the immunoglobulin heavy chain locus and genome-wide in activated B cells. DSBs from DNA replication stress and programmed rearrangement events often occur within the same cell and can recombine, driving the formation of chromosome rearrangements. In this R35 MIRA, we investigate how two common secondary structures formed in DNA—R loops and G quadruplexes—impact DSB formation, repair pathway choice, and chromosome rearrangement formation in primary, immortalized and transformed cells. My lab has developed novel methods to measure successful DNA repair at endogenous genomic loci in primary and immortalized cells, and to identify potential sites of transcription-associated replication stress. We have also defined the contribution of cell cycle progression and checkpoint activation on CR formation in both primary and immortalized cells. Here we will: 1) define molecular and genetic factors predisposing specific genomic loci to instability; and 2) functionally dissect the DNA repair pathways driving mutagenic chromosome rearrangement formation. We will use genetic, molecular, cell biological and genomic approaches to study complex rearrangement formation at the single cell and population level, tracking the impact on viability, cell fate, and further genome evolution. This R35 will define the repair pathways driving CR formation, the effect of CR formation on cell survival and cell tetraploidization, and the specific mutagenic signatures associated with G quadruplex stabilization and defective R loop metabolism.