The linear chromosome of the bacterium Streptomyces exhibits a remarkable genetic organization with grossly a central conserved region flanked by variable chromosomal arms. The terminal diversity co-locates with an intense DNA plasticity including the occurrence of large deletions associated to circularization and chromosomal arm exchange. These observations prompted us to assess the role of double strand break (DSB) repair in chromosome plasticity following. For that purpose, DSBs were induced along the chromosome using the meganuclease I-SceI. DSB repair in the central region of the chromosome was mutagenic at the healing site but kept intact the whole genome structure. In contrast, DSB repair in the chromosomal arms was mostly associated to the loss of the targeted chromosomal arm and extensive deletions beyond the cleavage sites. While homologous recombination occurring between copies of DNA sequences accounted for the most part of the chromosome rescue events, Non Homologous End Joining was involved in mutagenic repair as well as in huge genome rearrangements (i.e. circularization). Further, NHEJ repair was concomitant with the integration of genetic material at the healing site. We postulate that DSB repair drives genome plasticity and evolution in Streptomyces and that NHEJ may foster horizontal transfer in the environment.

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.

bHomologous recombination is a crucial mechanism that repairs a wide range of DNA lesions, including the most deleterious ones, double-strand breaks (DSBs). This multistep process is initiated by the resection of the broken DNA ends by a multisubunit helicase-nuclease complex exemplified by Escherichia coli RecBCD, Bacillus subtilis AddAB, and newly discovered Mycobacterium tuberculosis AdnAB. Here we show that in Streptomyces, neither recBCD nor addAB homologues could be detected. The only putative helicase-nuclease-encoding genes identified were homologous to M. tuberculosis adnAB genes. These genes are conserved as a single copy in all sequenced genomes of Streptomyces. The disruption of adnAB in Streptomyces ambofaciens and Streptomyces coelicolor could not be achieved unless an ectopic copy was provided, indicating that adnAB is essential for growth. Both adnA and adnB genes were shown to be inducible in response to DNA damage (mitomycin C) and to be independently transcribed. Introduction of S. ambofaciens adnAB genes in an E. coli recB mutant restored viability and resistance to UV light, suggesting that Streptomyces AdnAB could be a functional homologue of RecBCD and be involved in DNA damage resistance. Cells are under constant genotoxic pressure from both endogenous and exogenous sources. DNA damage needs to be repaired to avoid the formation of deleterious mutations, abortion of replication, and lethal chromosomal breakage. Homologous recombination (HR) is a crucial mechanism that repairs a variety of DNA lesions, including DNA double-strand breaks (DSBs), single-strand DNA gaps, and interstrand cross-links.DSBs are probably the most deleterious DNA damage that a cell can encounter. They are induced in cells by physical agents such as ionizing radiation or UV light, chemical agents, and natural products such as mitomycin C (MMC) or bleomycin. DSBs also result from replication fork collapse during chromosomal replication (1). The failure to repair DSBs can lead to cell death and, in the case of disrepair, can trigger large-scale chromosome rearrangements, favoring the generation of genetic diversity.In bacteria, DSBs are for the most part processed through HR, which requires a homologous DNA template to carry out faithful repair of the damaged DNA duplex. In intensively replicating cells (vegetative growth phase) or immediately after the passing of the replication fork, the sister chromatid can be used as an intact template. In nonreplicating phases, such as late stationary phase or in spores (which contain a single copy of the chromosome), DSBs are more likely repaired by an illegitimate repair pathway. Indeed, illegitimate recombination (IR) does not require an intact homologous sequence but as a consequence has reduced fidelity.The involvement of DSB repair by HR in a range of fundamental cellular processes (e.g., chromosome integrity, replication, segregation, etc.) reveals that HR is conserved in all living organisms. The initiating step of the repair mechanism consists of the resection of the DSB...