Double-strand break repair and G4 DNA stability in Caenorhabditis elegans

Abstract

DNA double-strand breaks (DSBs) can be repaired by three canonical repair pathways. Homologous recombination (HR) uses the sister chromatid or homologous chromosome as a template to repair the DSB in an error-free manner. In non-homologous end-joining (NHEJ), the broken ends are ligated with little or no sequence homology, and this is often accompanied by the loss of a few nucleotides. Single-strand annealing (SSA) uses sequence homology within the same chromosome and leads to deletion of one of the repeats and the intervening sequence. Using the model organism C. elegans, we study DSB repair in the context of a developing animal and in complex genetic backgrounds. We make use of a transgenic approach where the restriction enzyme I-SceI can be expressed in an inducible manner, combined with a reporter transgene that contains the 18-nt recognition site for I-SceI in an out-of-frame LacZ gene. In Chapter 2 of this thesis, we use this assay to reveal the activity of a fourth pathway, which we termed alternative end-joining (alt-EJ). This pathway seems to act as a backup for NHEJ because it predominates repair only in the absence of canonical NHEJ, but its repair products are characterized by frequent use of homology in a way that is similar to SSA. Alt-EJ operates independently of many known repair genes and leads to very efficient DSB repair even in triple mutants that are defective for HR, SSA and NHEJ. Despite its putative function as a backup for classic NHEJ, we show in Chapter 3 that, in contrast to NHEJ, alt-EJ only occurs in replicating cells, leading to DSB persistence in non-replicating NHEJ-deficient somatic cells. Although normally highly toxic, endogenous DSBs are introduced in a regulated manner in meiotic cells in the germline. These DSBs need to be repaired by HR to establish crossover formation between homologous chromosomes which is required for genetic diversity among the offspring and for correct chromosome segregation. In Chapter 4 we show that besides HR, other repair pathways are also active in the germline. Moreover, the response to DSBs is highly dependent on the stage of the cell cycle at the time of DSB induction and differs between different germline zones. Quadruplex of G4 DNA is a stable secondary ssDNA structure that can form in particular G-rich sequences during DNA replication. In mutants for the gene dog-1 (mammalian FANCJ), spontaneous deletions arise at G4 DNA. These deletions always initiate immediately downstream of the G-rich sequence and end at various locations downstream. In Chapter 5, we show that these deletions are likely formed through DSB intermediates, because they resemble DSBs at other locations in many ways. Remarkably, these DSBs are not repaired by one of the canonical repair routes, but may instead be repaired by alt-EJ or another mechanism. In Chapter 6, we show how deletion formation in dog-1 mutant background can be used a tool to isolate deletion alleles of many C. elegans genes.