Helicase Q promotes homology-driven DNA double-strand break repair and prevents tandem duplications.

DNA double-strand breaks are a major threat to cellular survival and genetic integrity. In addition to high fidelity repair, three intrinsically mutagenic DNA break repair routes have been described, i.e. single-strand annealing (SSA), polymerase theta-mediated end-joining (TMEJ) and residual ill-defined microhomology-mediated end-joining (MMEJ) activity. Here, we identify C. elegans Helicase Q (HELQ-1) as being essential for MMEJ as well as for SSA. We also find HELQ-1 to be crucial for the synthesis-dependent strand annealing (SDSA) mode of homologous recombination (HR). Loss of HELQ-1 leads to increased genome instability: patchwork insertions arise at deletion junctions due to abortive rounds of polymerase theta activity, and tandem duplications spontaneously accumulate in genomes of helq-1 mutant animals as a result of TMEJ of abrogated HR intermediates. Our work thus implicates HELQ activity for all DSB repair modes guided by complementary base pairs and provides mechanistic insight into mutational signatures common in HR-defective cancers.

Isolation and genetic characterisation of the Drosophila homologue of (SCE)REV3, encoding the catalytic subunit of DNA polymerase zeta.

In Drosophila, about 30 mutants are known that show hypersensitivity to the methylating agent methyl methane sulfonate (MMS). Addition of this agent to the medium results in an increased larval mortality of the mutants. Using a P-insertion mutagenesis screen, three MMS-sensitive mutants on chromosome II were isolated. One of these is allelic to the known EMS-induced mus205 (mutagen sensitive) mutant. In the newly isolated mutant, a P-element is detected in region 43E by in situ hybridisation. The localisation of mus205 to this region was confirmed by deficiency mapping. The gene was cloned and shows strong homology to the Saccharomyces cerevisiae REV3 gene. The REV3 gene encodes the catalytic subunit of DNA polymerase zeta, involved in translesion synthesis. The P-element is inserted in the first exon of the mus205 gene resulting in an aberrant mRNA, encoding a putative truncated protein containing only the first 13 of the 2130 aa native Drosophila protein. The mus205 mutant is hypersensitive to alkylating agents and UV, but not to ionising radiation. In contrast to reported data, in germ cells, the mutant has no effect on mutability by X-rays, NQO and alkylating agents. In somatic cells, the mutant shows no effect on MMS-induced mutations and recombinations. This phenotype of the Drosophila mus205 mutant is strikingly different from the phenotype of the yeast rev3 mutant, which is hypomutable after UV, X-rays, NQO and alkylating agents.

Molecular cloning, expression and chromosomal localisation of the mouse Rev3l gene, encoding the catalytic subunit of polymerase zeta.

The REV3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta which is involved in translesion synthesis. The mouse homolog of this gene, Rev3l, was cloned and sequenced. The gene encodes a putative protein of 3122 amino acids. The sequence conservation to its yeast counterpart is restricted to several regions. In the carboxy-terminal part of the protein all six domains are present that are characteristic for alpha-type DNA polymerases. In the amino-terminal part of the protein two regions can be identified with considerable similarity to the NT boxes of mouse polymerase delta. In addition, a region of 60 residues unique for the REV3 homologs can be found in the middle part of the protein. The mouse REV3L protein shows strong sequence conservation with the recently cloned human REV3L protein (86% identity overall). Northern blot analysis of various tissues of the mouse revealed that transcription of the Rev3l gene was highest in brain, ovaries and testis. The human REV3L gene was localised to the long arm of chromosome 6, region 21-22. The mouse equivalent maps to chromosome 10, distal to the c-myb gene, close to the Macs gene.