In vivo damage and recA-dependent repair of plasmid and chromosomal DNA in the radiation-resistant bacterium Deinococcus radiodurans

Abstract

Deinococcus radiodurans Rl and other members of this genus share extraordinary resistance to the lethal and mutagenic effects of ionizing radiation. We have recently identified a RecA homolog in strain RI and have shown that mutation of the corresponding gene causes marked radiosensitivity. We show here that following high-level exposure to gamma irradiation (1.75 megarads, the dose required to yield 37% of CFU for plateau-phase wild-type R1), the wild-type strain repairs >150 double-strand breaks per chromosome, whereas a recA-defective mutant (rec30) repairs very few or none. A heterologous Escherichia coli-D. radiodurans shuttle plasmid (pMD68) was constructed and found to be retained in surviving D. radiodurans Rl and rec30 following any radiation exposure up to the highest dose tested, 3 megarads. Plasmid repair was monitored in vivo following irradiation with 1.75 megarads in both R1/pMD68 and rec30/pMD68. Immediately after irradiation, plasmids from both strains contained numerous breaks and failed to transform E. coli. While irradiation with 1.75 megarads was lethal to rec30 cultures, a small amount of supercoiled plasmid was regenerated, but it lacked the ability to transform E. coli. In contrast, wild-type cultures showed a cell division arrest of about 10 h, followed by exponential growth. Supercoiled plasmid was regenerated at normal levels, and it readily transformed E. coli. These studies show that D. radiodurans retains a heterologous plasmid following irradiation and repairs it with the same high efficiency as its chromosomal DNA, while the repair defect in rec30 prevents repair of the plasmid. Taken together, the results of this study suggest that plasmid DNA damaged in vivo in D. radiodurans is repaired by recA-dependent mechanisms similar to those employed in the repair of chromosomal DNA.

Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation

“…After a dose of 15 kGy, DEIRA recovery typically progresses through three phases (5, 9, 10, 21): (i) early phase (0-3 h), where cell growth is inhibited and recA is induced, but there is little evidence of DNA repair; (ii) mid phase (3-9 h), where growth inhibition and recA expression continue, but with progressive DNA repair; and (iii) late phase (9-24 h), where recA is repressed and cell growth is restored. Consistent with these reports (5,6,(9)(10)(11), after the exposure of DEIRA to 15 kGy, Ϸ150 DSBs per haploid genome were inflicted (data not shown). As expected, after the 9-h lag in growth, cells grew exponentially and reached stationary phase 15 h later (Fig.…”

“…We examined changes in genomic expression in DEIRA recovering from an acute exposure to 15 kGy, which is about its D 37 (irradiation dose yielding 37% survival) under our standard conditions (5,(9)(10)(11). During the early and mid phases of recovery, no cell growth was observed, but within this interval many genes of diverse functional groups were induced (Table 3).…”

“…Within this large pool of significantly expressed genes, we operationally identified a subgroup that consists of genes with substantially greater expression levels and likely includes key players in the irradiation response. Table 3 lists genes and operons with recA-like expression patterns; DEIRA recA is critical to genomic restoration after irradiation (9)(10)(11)(12), and its induction is considered a dominant marker for the onset of homologous recombination (ref. 21; Fig.…”

“…Because most organisms, generally, can tolerate so few DSBs (8), radiationinduced DSBs and their repair have been difficult to study. In DEIRA, however, there are so many DSBs in fully viable irradiated cells after high-dose irradiation that the steps in DSB repair can be monitored directly in mass culture (5,(9)(10)(11). This characteristic has been exploited and used to examine the timing of DNA recombination (5, 10, 12) after high-dose irradiation and has revealed the sequential action of RecA-independent and -dependent pathways during repair (11).…”

Repair of extensive ionizing-radiation DNA damage at 95 degrees C in the hyperthermophilic archaeon Pyrococcus furiosus

“…Moreover, irradiation at lower doses, 1,000 and 2,000 Gy, which did not cause any loss of viability, displayed chromosome fragmentation (results not shown). In addition, the DSB repair process in P. furiosus did not require the dilution of the cells to a suitable density for growth, as in D. radiodurans (7,18).…”