Homology-directed repair is required for the development of radioresistance during S phase: interplay between double-strand break repair and checkpoint response

The S-phase-dependent radioresistance to killing uniformly seen in eukaryotic cells is absent in radiosensitive mutants with defects in genes involved in the repair of DNA double-strand breaks (DSBs) by homologous recombination (homologous recombination repair: HRR). This implicates, for the first time, a concrete DNA repair process in the radiosensitivity of a specific cell cycle phase. The cell cycle-dependent fluctuations in radiosensitivity reflect a fundamental and well-documented radiobiological phenomenon that still awaits a detailed molecular characterization. The underlying mechanisms are likely to combine aspects of DNA repair and cell cycle regulation. Advances in both fields allow a first dissection in the cell cycle of the molecular interplay between DSB repair and DNA damage checkpoint response and its contribution to cell survival. Here we review the available literature on the topic, speculate on the ramifications of this information for our understanding of cellular responses to DNA damage, and discuss future directions in research. An effort is made to integrate relevant phenomena of radiation action, such as low-dose radiosensitivity and the G(2) assay in this scheme.     

A constitutively expressed, truncated umuDC operon regulates the recA-dependent DNA damage induction of a gene in Acinetobacter baylyi strain ADP1

In response to environmentally caused DNA damage, SOS genes are up-regulated due to RecA-mediated relief of LexA repression. In Escherichia coli, the SOS umuDC operon is required for DNA damage checkpoint functions and for replicating damaged DNA in the error-prone process called SOS mutagenesis. In the model soil bacterium Acinetobacter baylyi strain ADP1, however, the content, regulation, and function of the umuDC operon are unusual. The umuC gene is incomplete, and a remnant of an ISEhe3-like transposase has replaced the middle 57% of the umuC coding region. The umuD open reading frame is intact, but it is 1.5 times the size of other umuD genes and has an extra 5' region that lacks homology to known umuD genes. Analysis of a umuD::lacZ fusion showed that umuD was expressed at very high levels in both the absence and presence of mitomycin C and that this expression was not affected in a recA-deficient background. The umuD mutation did not affect the growth rate or survival after UV-induced DNA damage. However, the UmuD-like protein found in ADP1 (UmuDAb) was required for induction of an adjacent DNA damage-inducible gene, ddrR. The umuD mutation specifically reduced the DNA damage induction of the RecA-dependent DNA damage-inducible ddrR locus by 83% (from 12.9-fold to 2.3-fold induction), but it did not affect the 33.9-fold induction of benA, an unrelated benzoate degradation gene. These data suggest that the response of the ADP1 umuDC operon to DNA damage is unusual and that UmuDAb specifically regulates the expression of at least one DNA damage-inducible gene.     

ClpR protein-like regulator specifically recognizes RecA protein-independent promoter motif and broadly regulates expression of DNA damage-inducible genes in mycobacteria

The RecA-dependent DNA damage response pathway (SOS response) appears to be the major DNA repair mechanism in most bacteria, but it has been suggested that a RecA-independent mechanism is responsible for controlling expression of most damage-inducible DNA repair genes in Mycobacterium tuberculosis. The specific reparative responses and molecular mediators involved in the DNA repair mechanism remain largely unclear in this pathogen and its related species. In this study, a mycobacterial ClpR-like regulator, corresponding to Rv2745c in M. tuberculosis and to Ms2694 in M. smegmatis mc(2)155, was found to interact with the promoter regions of multiple damage-inducible DNA repair genes. Specific binding of the ClpR-like factor to the conserved RecA-independent promoter RecA-NDp motif was then confirmed using in vitro electrophoretic mobility shift assays as well as in vivo chromatin immunoprecipitation experiments. The ClpR knock-out experiments, in combination with quantitative real time PCR assays, demonstrated that the expression of these RecA-independent genes were significantly down-regulated in the mutant strain of M. smegmatis in response to a DNA-damaging agent compared with the wild type strain. Furthermore, the ClpR-like factor was shown to contribute to mycobacterial genomic stability. These results enhance our understanding of the function of the ClpR regulator and the regulatory mechanism of RecA-independent DNA repair in mycobacteria.     

Cloning and characterization of DNA damage-inducible promoter regions from Bacillus subtilis.

DNA damage-inducible (din) genes in Bacillus subtilis are coordinately regulated and together compose a global regulatory network that has been termed the SOS-like or SOB regulon. To elucidate the mechanisms of SOB regulation, operator/promoter regions from three din loci (dinA, dinB, and dinC) of B. subtilis were cloned. Operon fusions constructed with these cloned din promoter regions rendered reporter genes damage inducible in B. subtilis. Induction of all three din promoters was dependent upon a functional RecA protein. Analysis of these fusions has localized sequences required for damage-inducible expression of the dinA, dinB, and dinC promoters to within 120-, 462-, and 139-bp regions, respectively. Comparison of the nucleotide sequences of these three din promoters with the recA promoter, as well as with the promoters of other loci associated with DNA repair in B. subtilis, has identified the consensus sequence GAAC-N4-GTTC as a putative SOB operator site.