Role of DNA Repair by Nonhomologous-End Joining in Bacillus subtilis Spore Resistance to Extreme Dryness, Mono- and Polychromatic UV, and Ionizing Radiation

Abstract

The role of DNA repair by nonhomologous-end joining (NHEJ) in spore resistance to UV, ionizing radiation, and ultrahigh vacuum was studied in wild-type and DNA repair mutants (recA, splB, ykoU, ykoV, and ykoU ykoV mutants) of Bacillus subtilis. NHEJ-defective spores with mutations in ykoU, ykoV, and ykoU ykoV were significantly more sensitive to UV, ionizing radiation, and ultrahigh vacuum than wild-type spores, indicating that NHEJ provides an important pathway during spore germination for repair of DNA double-strand breaks.It has been shown that endospores of gram-positive bacteria can remain viable for at least thousands of years (5, 44, 54; reviewed in reference 31). Bacterial spores persist in a metabolically inactive state, and environmental damage to spore cellular components accumulates unrepaired until germination and outgrowth (32). However, Bacillus subtilis spores are highly resistant to different environmental stresses, such as toxic chemicals and biocidal agents, desiccation, pressure and temperature extremes, and ionizing and UV radiation. The reason for this high resistance to environmental extremes lies partly in the spore structure itself: spores possess thick layers of highly cross-linked coat proteins (13), a modified peptidoglycan spore cortex, and abundant intracellular constituents such as the calcium chelate of dipicolinic acid and small, acidsoluble spore proteins (␣/␤-type SASP), as protectants of spore DNA (46, 50). Binding of ␣/␤-type SASP to spore DNA, coupled with spore core dehydration, appears to change the helical conformation of spore DNA from the B form to an A-like form (34, 48), which in turn alters its UV photochemistry to favor the production of 5-thyminyl-5,6-dihydrothymine, the unique spore-specific spore photoproduct (SP) (8,32,35,50). For the removal of the SP, spores possess an SP-specific repair enzyme called SP lyase, encoded by the splB gene, that monomerizes the SP dimer back to two thymine residues in an adenosyl-radical-dependent reaction (4,28,42).While the UV photochemistry of spore DNA and the repair of UV damage to DNA during germination are well described (12,32,33,47,50), there has been relatively little work on the nature of DNA damage in spores caused by ionizing radiation or extreme dryness and on the occurrence of a specific DNA repair system(s) for repair of this damage. It is assumed that DNA double-strand breaks (DSB), which are the most critical damage caused by ionizing radiation (57) and desiccation (9, 10, 39) in vegetative cells, are also induced in bacterial spores. Spores of B. subtilis contain a single chromosome arranged in a toroidal shape (16, 41); therefore, the homologous recombination pathway, which requires at least two homologous chromosomes, cannot operate on DSB during spore germination (55). An alternative repair pathway for DSB induced in spore DNA, nonhomologous-end joining (NHEJ) (3, 56), is considered here. This pathway as it occurs in eukaryotic cells requires a DNA end-binding component called Ku (Ku70 and Ku80) (58). The fir...

Sub-THz Vibrational Spectroscopy of Bacterial Cells and Molecular Components

“…Specifically, additional absorption peaks are revealed at 13.7, 15.3, 21.2 and 22 cm -1 for the sample in the vacuum, while the peak at 11.5 cm -1 (in the air) d isappeared in the vacuum. Also the maximu m at 17.1 cm -1 observed for the sample in air shifts to 16.7 cm -1 in vacuu m. It has been documented that the spores can survive extreme desiccation fo r extended periods of time without any serious side effects [37,38]. In our experiments, spores were exposed to a low vacuum for less than 20 minutes.…”

Space Microbiology

“…Spores of the triple mutant repair-deficient strain TKJ 8431 (uvrA10 ssp-1 recA1) of B. subtilis, which are deficient in recombination repair, were the most sensitive specimens under conditions of space vacuum (115). Using space simulation facilities, Moeller et al (170) showed that NHEJ is a highly efficient repair pathway for DNA DSBs induced in spores of B. subtilis by high vacuum. They hypothesize that NHEJ is a key strategy used during spore germination to repair DSBs caused by ultra-high-vacuuminduced extreme desiccation, as well as by other extreme factors, such as UV and ionizing radiation, encountered during prolonged exposure to the harsh environment of space.…”

“…These include rejoining the broken ends, by homologous recombination with a sister strand molecule (71) or by nonhomologous end joining (NHEJ) (22). In spores of B. subtilis, which contain a single chromosome arranged in a toroidal shape (70), NHEJ is the most efficient repair pathway during germination of spores exposed to ionizing radiation, such as X-rays (170) or HZE particles (173).…”

Characterization of the Mycobacterial AdnAB DNA Motor Provides Insights into the Evolution of Bacterial Motor-Nuclease Machines

“…By contrast, NHEJ protects the bacterial chromosome against DSBs during quiescent states, when there is no sister chromatid available to direct HR (12,13). The recent finding that dormant mycobacteria develop into spores containing a single chromosome (14) suggests a role for NHEJ in spore viability under conditions of clastogenic stress, as demonstrated for the classic spore-forming bacterium, Bacillus subtilis (15,16). The mycobacterial NHEJ mechanism, unlike that of HR, is conspicuously mutagenic (9,11).…”