Multiple and Variable NHEJ-Like Genes Are Involved in Resistance to DNA Damage in Streptomyces ambofaciens

Abstract

Non-homologous end-joining (NHEJ) is a double strand break (DSB) repair pathway which does not require any homologous template and can ligate two DNA ends together. The basic bacterial NHEJ machinery involves two partners: the Ku protein, a DNA end binding protein for DSB recognition and the multifunctional LigD protein composed a ligase, a nuclease and a polymerase domain, for end processing and ligation of the broken ends. <i>In silico</i> analyses performed in the 38 sequenced genomes of <i>Streptomyces</i> species revealed the existence of a large panel of NHEJ-like genes. Indeed, <i>ku</i> genes or <i>ligD</i> domain homologues are scattered throughout the genome in multiple copies and can be distinguished in two categories: the "core" NHEJ gene set constituted of conserved loci and the "variable" NHEJ gene set constituted of NHEJ-like genes present in only a part of the species. In <i>Streptomyces ambofaciens</i> ATCC23877, not only the deletion of "core" genes but also that of "variable" genes led to an increased sensitivity to DNA damage induced by electron beam irradiation. Multiple mutants of <i>ku</i>, ligase or polymerase encoding genes showed an aggravated phenotype compared to single mutants. Biochemical assays revealed the ability of Ku-like proteins to protect and to stimulate ligation of DNA ends. RT-qPCR and GFP fusion experiments suggested that <i>ku</i>-like genes show a growth phase dependent expression profile consistent with their involvement in DNA repair during spores formation and/or germination.