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. In silico analyses performed in the 38 sequenced genomes of Streptomyces species revealed the existence of a large panel of NHEJ-like genes. Indeed, ku genes or ligD 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 Streptomyces ambofaciens 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 ku, 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 ku-like genes show a growth phase dependent expression profile consistent with their involvement in DNA repair during spores formation and/or germination.

Bacterial NHEJ: a never ending story

“…♦ in M. tuberculosis denotes accessory actors involved in NHEJ, based on studies of their homologs in M. smegmatis coding for different LigD with mutated domain (see text). In S. ambofaciens , accessory actors involved in response to DNA damage are highlighted by an asterisk (Hoff et al , ; ). For the sake of clarity, only one dimer of Ku is shown at DNA ends and the ability of Ku to thread inward the DNA molecule has not been represented.…”

“…Among the bacterial species predicted to possess a NHEJ repair system, some actually encode several putative Ku homologs, mainly actinobacteria of the streptomycetes family, and proteobacteria of the α subdivision (McGovern et al , ). For instance, more than 70% of Streptomyces species encode more than one ku gene (up to 4 in Streptomyces coelicolor ) (Hoff et al , ), and more than half of α‐proteobacteria possessing a ku gene actually have two or more (up to 6 in Rhizobium leguminosarum bv trifolii ). Some of the ku genes are located on plasmids, suggesting their putative acquisition via horizontal gene transfer.…”

“…The variable set of Streptomyces NHEJ‐like genes also includes kuB and kuC , the latter often being plasmid‐borne and often laying in the close vicinity of ligD . Although they are not conserved, these genes take part in DNA repair since their mutation in Streptomyces ambofaciens confers spore sensitivity to IR and increases the sensitivity of the core gene set mutants (Hoff et al , ). In S. ambofaciens, the involvement of KuA in a NHEJ system has been demonstrated together with the existence of a secondary illegitimate DSB repair system that could imply these variable genes (Hoff et al , ).…”

“…In this regard, Streptomyces is not an exception since M. smegmatis (see above and below, PolD1, PolD2) and numerous eukaryotes, especially vertebrates, also possess several polymerases involved in NHEJ (Ramsden and Asagoshi, ). A first hypothesis to explain this observation is based on the difference in expression profile: indeed, S. ambofaciens polR seems constitutively expressed, whereas polK transcription was not detected in the tested conditions (Hoff et al , ), but might be induced in response to some genotoxic stress. Alternatively or concomitantly, these PolDoms could display different template requirements, as for the four polymerases of the X family found in vertebrates (Polβ, Polλ, Polµ and TdT, for review see Ramsden and Asagoshi, ).…”

“…The different functions of this extended C‐ter domain raise the question about the efficiency of the NHEJ in bacteria encoding a Ku without this domain and/or about the role of Ku and its extended C‐ter in another DNA repair mechanism (for instance the BER process, see below). In Streptomyces , one of the accessory Ku (KuB), present in only 8 of 38 species (Hoff et al , ), possesses an extra C‐ter domain weakly related to the RhoN domain (Pfam 07498) which role in Ku protein remains to be elucidated.…”