DNA double strand breaks (DSBs) are among the most toxic lesions that can occur in the genome, and at least three distinct repair pathways can be used for the repair of these breaks. These are homologous recombination (HR), single-strand annealing (SSA) and non-homologous end-joining (NHEJ). The choice of which pathway to be used depends, among others, on cell type, availability of necessary substrates and stage of the cell cycle. It has so far not been possible to study repair of a targeted DSB in the context of a developing animal, and therefore, the relative contribution of each of these pathways has remained unclear. Also, new genes remain to be identified. We have made transgenic C. elegans strains that contitutively or inducibly express the yeast rare-cutting endonuclease I-SceI and that also carry a reporter LacZ-gene, interrupted by the 18-nt I-SceI recognition sequence. Repair of I-SceI-induced DSBs can be monitored by restoration of LacZ-expression, which we think will predominantly result from repair by the SSA pathway due to the presence of homologous sequences before and after the breaksite. This is supported by our observation that knockdown of the HR components
rad-51 and
brc-1 increase LacZ expression in this assay, suggesting a shift from HR towards SSA, whereas RNAi of the ssDNA-binding protein M04F3.1 (RPA2) decreases expression. We are currently testing a set of 40 candidate genes, that resulted in ionizing radiation sensitivity when knocked down by RNAi, for a role in DSB repair using this assay. We are also developing variations on this assay to study other types of DNA repair. We have employed similar LacZ-based assays in forward genetic screens to find genes that are involved in stabilization of repeated sequences in the genome. In one approach, we placed a stretch of guanines, 3 flanked by a nonsense codon, in the LacZ-sequence and clonally screened EMS-mutagenized animals for reversion of LacZ expression. Although we did not find any new genes involved in the maintenance of G-stretches, we did identify 5 new knockout alleles of the known stabilizer of G-tracts
dog-11. To characterize the DNA substrate for
dog-1, we hybridized DNA from inbred
dog-1 mutants to a custom-made tiling array and found that sequences predicted to form quadruplex (G4 DNA) structures were unstable in these mutants. We are now investigating whether these structures form physical blocks to DNA replication forks by purification of Okazaki fragments followed by qPCRs analyses. 1. Cheung I. et al, Disruption of
dog-1 in Caenorhabditis elegans triggers deletions upstream of guanine-rich DNA, Nat Genet. 2002, 31(4):405-9.