Shigeru Kitayama

PprI : a general switch responsible for extreme radioresistance of Deinococcus radiodurans

Deinococcus radiodurans exhibits an extraordinary ability to withstand the lethal and mutagenic effects of DNA damaging agents, particularly, ionizing radiation. Available evidence indicates that efficient repair of DNA damage and protection of the chromosomal structure are mainly responsible for the radioresistance. Little is known about the biochemical basis for this phenomenon. We have identified a unique gene, pprI, as a general switch for downstream DNA repair and protection pathways, from a natural mutant, in which pprI is disrupted by a transposon. Complete functional disruption of the gene in wild-type leads to dramatic sensitivity to ionizing radiation. Radioresistance of the disruptant could be fully restored by complementation with pprI. In response to radiation stress, PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection pathways in response to radiation stress.

PprA: a novel protein from Deinococcus radiodurans that stimulates DNA ligation.

The extraordinary radiation resistance of Deinococcus radiodurans results from the efficient capacity of the bacterium to repair DNA double‐strand breaks. By analysing the DNA damage repair‐deficient mutant, KH311, a unique radiation‐inducible gene (designated pprA) responsible for loss of radiation resistance was identified. Investigations in vitro showed that the gene product of pprA (PprA) preferentially bound to double‐stranded DNA carrying strand breaks, inhibited Escherichia coli exonuclease III activity, and stimulated the DNA end‐joining reaction catalysed by ATP‐dependent and NAD‐dependent DNA ligases. These results suggest that D. radiodurans has a radiation‐induced non‐homologous end‐joining repair mechanism in which PprA plays a critical role.

Possibility of the repair of double-strand scissions in Micrococous radiodurans DNA caused by gamma-rays

Abstract A substantial variance in the ability to repair radiation damage to DNA has been found among various types of bacteria, resulting in different radiosensitivities. For instance, increase in sedimentation rates of alkali-denatured DNA of irradiated cells during reincubation after exposure to X-rays, which is interpreted as the repair of single-strand scissions in DNA, has been observed for E. coli , B r , a resistant strain, but not for E. coli Bs−1, a sensitive strain( McGrath and Williams, 1966 ). However, most bacteria are assumed to be unable to repair double-strand scissions and Kaplan(1966) has presented an evidence for this view with E. coli K12 by sedimentation analysis on neutral sucrose gradients. On the other hand, Dean et al (1966) have suggested the capacity of M. radiodurans , which is a vegetative bacterium extremely resistant to ionizing and ultraviolet radiations, for repairing double-strand scissions in DNA. The purpose of the present paper is to examine this possibility by the sedimentation analysis of tritiated materials in M. radiodurans after γ-ray irradiation.

The Deinococcus radiodurans uvrA gene: identification of mutation sites in two mitomycin-sensitive strains and the first discovery of insertion sequence element from deinobacteria

Deinococcus radiodurans (Dr) possesses a prominent ability to repair the DNA injury induced by various DNA-damaging agents including mitomycin C (MC), ultraviolet light (UV) and ionizing radiation. DNA damage resistance was restored in MC sensitive (MC(S)) mutants 2621 and 3021 by transforming with DNAs of four cosmid clones derived from the gene library of strain KD8301, which showed wild type (wt) phenotype to DNA-damaging agents. Gene affected by mutation (mtcA or mtcB) in both mutants was cloned and its nucleotide (nt) sequence was determined. The deduced amino acid (aa) sequence of the gene product consists of 1016 aa and shares homology with many bacterial UvrA proteins. The mutation sites of both mutants were identified by analyzing the polymerase chain reaction (PCR) fragments derived from the genomic DNA of the mutants. A 144-base pair (bp) deletion including the start codon for the uvrA gene was observed in DNA of the mutant 3021, causing a defect in the gene. On the other hand, an insertion sequence (IS) element intervened in the uvrA gene of the mutant 2621, suggesting the insertional inactivation of the gene. The IS element comprises 1322-bp long, flanked by 19-bp inverted terminal repeats (ITR), and generated a 6-bp target duplication (TD). Two open reading frames (ORFs) were found in the IS element. The deduced aa sequences of large and small ORFs show homology to a putative transposase found in IS4 of Escherichia coli (Ec) and to a resolvase found in ISXc5 of Xanthomonas campestris (Xc), respectively. This is the first discovery of IS element in deinobacteria, and the IS element was designated IS2621.

Changes in cellular proteins ofDeinococcus radiodurans followingγ-irradiation

In order to examine radiation-induced proteins in an extremely radioresistant bacterium,Deinococcus radiodurans R1, changes in cellular proteins after γ-irradiation were analysed by two-dimensional gel electrophoresis and silver staining. Nine proteins (190, 120, 87, 60, 58, 52, 46, 41 and 41 kDa) were increased (or appeared) and more than 13 proteins diminished after γ-irradiation at 6 kGy. Increase of eight proteins (except for 190-kDa protein) was prevented when the cells were irradiated in the presence of chloramphenicol. Three proteins, 87, 60 and 46 kDa, continued to be synthesized during post-irradiation incubation, and the amounts of these proteins increased with higher doses in a range of 1–12 kGy. Changes in the amount of proteins after irradiation in theR1 strain were compared with those in a moderately radioresistant mutant (rec 1) and in a highly radiosensitive mutant (rec30). These three proteins were increased in bothR1 andrec 1, but not inrec 30, suggesting that they are characteristic for radioresistant strains. In addition, from the microsequence analysis, the 46-kDa protein was found to be homologous to the EF-Tu protein ofEscherichia coli, whereas the remarkable homologous sequence to the N-terminal of the 60-kDa protein was not found among the known proteins.

Mechanism for Radiation Lethality in M. Radiodurans

SummaryAfter irradiation with doses which yield almost 100 per cent survival using ordinary agar plating methods, the cells of M. radiodurans lost their colony-forming ability when the post-irradiation incubation was carried out in the presence of inhibitors of protein or RNA synthesis. This result suggests the relative importance of protein and/or RNA synthesis as primary targets for the killing action of radiation.

Mutation of D. radiodurans in a gene homologous to ruvB of E. coli.

Following the digestion of chromosomal DNA of Deinococcus radiodurans with a restriction enzyme a partial genomic library was constructed using lambda phage as a vector. A phage clone whose DNA can complement the deficiency in a radiation-sensitive mutant of D. radiodurans was isolated. Following the subcloning using phasmid vector, a hybrid plasmid containing 1.2 kb inserted DNA was obtained. After the determination of nucleotide sequence, the deduced amino acid sequence showed close homology to RuvB protein of Escherichia coli; approximately 81% of the amino acids (310 residues in total) was homologous (152 were identical and 100 amino acids were similar). The putative protein has a conserved ATP binding domain characteristic of DNA helicases. However, we could not find an SOS promoter and ORF for RuvA protein in the sequence upstream of ruvB in contrast to the E. coli homologue. The mutant was transformed with exogenous DNA at the same rate as the wild-type cells, but it was moderately sensitive to UV, gamma-rays and to interstrand cross-linking reagents.

Mutation in recR gene of Deinococcus radiodurans and possible involvement of its product in the repair of DNA interstrand cross-links

We previously reported that some Deinococcus radiodurans mutants are sensitive to DNA interstrand cross-linking agents but resistant to UV and gamma-rays. We isolated DNA fragments from a D. radiodurans genomic library which complemented the mitomycin C sensitivity of one of these mutants. One 3.2kb-long fragment contains an open reading frame of approximately 700bp and the deduced amino acid sequence is very homologous to other prokaryotic RecR proteins. This open reading frame in the mitomycin C-sensitive mutant strain contains a frame shift mutation at its carboxyl terminal region. These data suggest that RecR protein plays an important role in the resistance to interstrand cross-links in this bacterium.

Adaptive repair of cross-links in DNA of Micrococcus radiodurans

Cross-links in the DNA of Micrococcus radiodurans induced by mitomycin C were repaired during post-incubation. This repair process was inhibited in cells post-incubated in the presence of chloramphenicol. However, the removal of cross-links in DNA was almost normal, even in the presence of chloramphenicol, if the cells were pretreated with lower concentrations of mitomycin C.

Loss of characteristic radiation resistance by mutation of Micrococcus radiodurans

Abstract The exceptional radioresistance of M. radiodurans was lost by a mutation, and the isolated mutant, KH840, had almost the same radiosensitivity as E. coli K12 AB1157 or B/r. DNA-strand scissions in this mutant were rejoined partly during post-irradiation incubation. Reduced DNA polymerizing activity and extensive degradation of the product was observed in vitro in the crude extract from the sensitive strain. The lower repair capability is not accompanied by the reduced transformation frequency.