Impact of C-terminal domains of paralogous single-stranded DNA binding proteins from Streptomyces coelicolor on their biophysical properties and biological functions.

Single-stranded DNA-binding proteins (SSB) are crucial in DNA metabolism. While Escherichia coli SSB is extensively studied, the significance of its C-terminal domain has only recently emerged. This study explored the significance of C-domains of two paralogous Ssb proteins in S. coelicolor. Mutational analyses of C-domains uncovered a novel role of SsbA during sporulation-specific cell division and demonstrated that the C-tip is non-essential for survival. In vitro methods revealed altered biophysical and biochemical properties of Ssb proteins with modified C-domains. Determined hydrodynamic properties suggested that the C-domains of SsbA and SsbB occupy a globular position proposed to mediate cooperative binding. Only SsbA was found to form biomolecular condensates independent of the C-tip. Interestingly, the truncated C-domain of SsbA increased the molar enthalpy of unfolding. Additionally, calorimetric titrations revealed that C-domain mutations affected ssDNA binding. Moreover, this analysis showed that the SsbA C-tip aids binding most likely by regulating the position of the flexible C-domain. It also highlighted ssDNA-induced conformational mobility restrictions of all Ssb variants. Finally, the gel mobility shift assay confirmed that the intrinsically disordered linker is essential for cooperative binding of SsbA. These findings highlight the important role of the C-domain in the functioning of SsbA and SsbB proteins.

Chromosome Segregation and Cell Division Defects in Escherichia coli Recombination Mutants Exposed to Different DNA-Damaging Treatments.

Homologous recombination repairs potentially lethal DNA lesions such as double-strand DNA breaks (DSBs) and single-strand DNA gaps (SSGs). In Escherichia coli, DSB repair is initiated by the RecBCD enzyme that resects double-strand DNA ends and loads RecA recombinase to the emerging single-strand (ss) DNA tails. SSG repair is mediated by the RecFOR protein complex that loads RecA onto the ssDNA segment of gaped duplex. In both repair pathways, RecA catalyses reactions of homologous DNA pairing and strand exchange, while RuvABC complex and RecG helicase process recombination intermediates. In this work, we have characterised cytological changes in various recombination mutants of E. coli after three different DNA-damaging treatments: (i) expression of I-SceI endonuclease, (ii) γ-irradiation, and (iii) UV-irradiation. All three treatments caused severe chromosome segregation defects and DNA-less cell formation in the ruvABC, recG, and ruvABC recG mutants. After I-SceI expression and γ-irradiation, this phenotype was efficiently suppressed by the recB mutation, indicating that cytological defects result mostly from incomplete DSB repair. In UV-irradiated cells, the recB mutation abolished cytological defects of recG mutants and also partially suppressed the cytological defects of ruvABC recG mutants. However, neither recB nor recO mutation alone could suppress the cytological defects of UV-irradiated ruvABC mutants. The suppression was achieved only by simultaneous inactivation of the recB and recO genes. Cell survival and microscopic analysis suggest that chromosome segregation defects in UV-irradiated ruvABC mutants largely result from defective processing of stalled replication forks. The results of this study show that chromosome morphology is a valuable marker in genetic analyses of recombinational repair in E. coli.

Characterization of gross genome rearrangements in Deinococcus radiodurans recA mutants.

Genome stability in radioresistant bacterium Deinococcus radiodurans depends on RecA, the main bacterial recombinase. Without RecA, gross genome rearrangements occur during repair of DNA double-strand breaks. Long repeated (insertion) sequences have been identified as hot spots for ectopic recombination leading to genome rearrangements, and single-strand annealing (SSA) postulated to be the most likely mechanism involved in this process. Here, we have sequenced five isolates of D. radiodurans recA mutant carrying gross genome rearrangements to precisely characterize the rearrangements and to elucidate the underlying repair mechanism. The detected rearrangements consisted of large deletions in chromosome II in all the sequenced recA isolates. The mechanism behind these deletions clearly differs from the classical SSA; it utilized short (4-11 bp) repeats as opposed to insertion sequences or other long repeats. Moreover, it worked over larger linear DNA distances from those previously tested. Our data are most compatible with alternative end-joining, a recombination mechanism that operates in eukaryotes, but is also found in Escherichia coli. Additionally, despite the recA isolates being preselected for different rearrangement patterns, all identified deletions were found to overlap in a 35 kb genomic region. We weigh the evidence for mechanistic vs. adaptive reasons for this phenomenon.

RecBCD- RecFOR-independent pathway of homologous recombination in Escherichia coli.

The RecA protein is a key bacterial recombination enzyme that catalyzes pairing and strand exchange between homologous DNA duplexes. In Escherichia coli, RecA protein assembly on DNA is mediated either by the RecBCD or RecFOR protein complexes. Correspondingly, two recombination pathways, RecBCD and RecF (or RecFOR), are distinguished in E. coli. Inactivation of both pathways in recB(CD) recF(OR) mutants results in severe recombination deficiency. Here we describe a novel, RecBCD- RecFOR-independent (RecBFI) recombination pathway that is active in ΔrecBCD sbcB15 sbcC(D) ΔrecF(OR) mutants of E. coli. In transductional crosses, these mutants show only four-fold decrease of recombination frequency relative to the wild-type strain. At the same time they recombine 40- to 90-fold better than their sbcB+ sbcC+ and ΔsbcB sbcC counterparts. The RecBFI pathway strongly depends on recA, recJ and recQ gene functions, and moderately depends on recG and lexA functions. Inactivation of dinI, helD, recX, recN, radA, ruvABC and uvrD genes has a slight effect on RecBFI recombination. After exposure to UV and gamma irradiation, the ΔrecBCD sbcB15 sbcC ΔrecF mutants show moderately increased DNA repair proficiency relative to their sbcB+ sbcC+ and ΔsbcB sbcC counterparts. However, introduction of recA730 allele (encoding RecA protein with enhanced DNA binding properties) completely restores repair proficiency to ΔrecBCD sbcB15 sbcC ΔrecF mutants, but not to their sbcB+ sbcC+ and ΔsbcB sbcC derivatives. Fluorescence microscopy with UV-irradiated recA-gfp fusion mutants suggests that the kinetics of RecA filament formation might be slowed down in the RecBFI pathway. Inactivation of 3'-5' exonucleases ExoVII, ExoIX and ExoX cannot activate the RecBFI pathway in ΔrecBCD ΔsbcB sbcC ΔrecF mutants. Taken together, our results show that the product of the sbcB15 allele is crucial for RecBFI pathway. Besides protecting 3' overhangs, SbcB15 protein might play an additional, more active role in formation of the RecA filament.

RecF, UvrD, RecX and RecN proteins suppress DNA degradation at DNA double-strand breaks in Escherichia coli.

Double strand breaks (DSBs) in E. coli chromosome (such as those induced by gamma rays) are repaired by recombination repair, during which a certain amount of DNA gets degraded. We monitored DNA degradation in gamma-irradiated cells to assess processing of DSBs. DNA degradation in irradiated cells is regulated by RecA protein concentration and its affinity of ssDNA binding, as well as by exonucleases that trim 3'-terminated ss tails. Here we determined the effects of proteins that affect formation and stability of RecA nucleofilaments on DNA degradation and cell survival. RecF and UvrD suppressed DNA degradation through RecA protein function and SOS induction, while also improving gamma survival. RecF and UvrD function in one pathway. Acting along with RecF, RecX suppressed DNA degradation and stimulated gamma-survival, which also depends on RecA protein and SOS induction. Furthermore, we determined a role in DNA degradation of several proteins that participate in DSB repair. RecN was required for DNA repair and for degradation suppression, acting on the RecABCD pathway. Furthermore, we show that SSB protein overproduction did not affect DNA degradation. Inactivation of RecG and RuvABC, proteins that catalyze the postsynaptic phase of recombination repair of DSBs, also did not affect DNA degradation, suggesting that once formed, recombination intermediates are not subject to DNA degradation, and that the postsynaptic phase is an irreversible, single-round process, unlike the presynaptic phase, which is mostly repetitive.

Elevated Rate of Genome Rearrangements in Radiation-Resistant Bacteria.

A number of bacterial, archaeal, and eukaryotic species are known for their resistance to ionizing radiation. One of the challenges these species face is a potent environmental source of DNA double-strand breaks, potential drivers of genome structure evolution. Efficient and accurate DNA double-strand break repair systems have been demonstrated in several unrelated radiation-resistant species and are putative adaptations to the DNA damaging environment. Such adaptations are expected to compensate for the genome-destabilizing effect of environmental DNA damage and may be expected to result in a more conserved gene order in radiation-resistant species. However, here we show that rates of genome rearrangements, measured as loss of gene order conservation with time, are higher in radiation-resistant species in multiple, phylogenetically independent groups of bacteria. Comparison of indicators of selection for genome organization between radiation-resistant and phylogenetically matched, nonresistant species argues against tolerance to disruption of genome structure as a strategy for radiation resistance. Interestingly, an important mechanism affecting genome rearrangements in prokaryotes, the symmetrical inversions around the origin of DNA replication, shapes genome structure of both radiation-resistant and nonresistant species. In conclusion, the opposing effects of environmental DNA damage and DNA repair result in elevated rates of genome rearrangements in radiation-resistant bacteria.

RecQ helicase acts before RuvABC, RecG and XerC proteins during recombination in recBCD sbcBC mutants of Escherichia coli.

The RecQ helicase is required by the RecF recombination pathway that is operative in recBC(D) sbcB sbcC(D) mutants of Escherichia coli. Genetic data suggest that RecQ participates in resection of DNA ends during initiation of recombination. In vitro, RecQ can unwind a variety of DNA substrates, including recombination intermediates such as D-loops and Holliday junctions. However, its potential role in processing of recombination intermediates during the late stage of the RecF pathway has not been genetically tested. Here we studied the effect of a recQ mutation on transductional recombination and DNA repair after γ-irradiation in ΔrecBCD ΔsbcB sbcC strains deficient for RuvABC, RecG and XerC proteins. RuvABC and RecG proteins process recombination intermediates in the late stage of recombination, whereas XerC is required to resolve chromosome dimers formed upon recombination. Our results do not reveal any substantial synergistic effect between the recQ mutation, on one hand, and ruvABC, recG and xerC mutations on the other. In addition, the recQ mutation suppresses chromosome segregation defects in γ-irradiated ruvABC recG and xerC mutants. These results suggest that RecQ acts upstream of RuvABC, RecG and XerC proteins, a finding that is compatible with its primary role in initiation of the RecF recombination pathway.

Exonuclease VII is involved in "reckless" DNA degradation in UV-irradiated Escherichia coli.

The recA mutants of Escherichia coli exhibit an abnormal DNA degradation that starts at sites of double-strand DNA breaks (DSBs), and is mediated by RecBCD exonuclease (ExoV). This "reckless" DNA degradation occurs spontaneously in exponentially growing recA cells, and is stimulated by DNA-damaging agents. We have previously found that the xonA and sbcD mutations, which inactivate exonuclease I (ExoI) and SbcCD nuclease, respectively, markedly suppress "reckless" DNA degradation in UV-irradiated recA cells. In the present work, we show that inactivation of exonuclease VII (ExoVII) by an xseA mutation contributes to attenuation of DNA degradation in UV-irradiated recA mutants. The xseA mutation itself has only a weak effect, however, it acts synergistically with the xonA or sbcD mutations in suppressing "reckless" DNA degradation. The quadruple xseA xonA sbcD recA mutants show no sign of DNA degradation during post-irradiation incubation, suggesting that ExoVII, together with ExoI and SbcCD, plays a crucial role in regulating RecBCD-catalyzed chromosome degradation. We propose that these nucleases act on DSBs to create blunt DNA ends, the preferred substrates for the RecBCD enzyme. In addition, our results show that in UV-irradiated recF recA(+) cells, the xseA, xonA, and sbcD mutations do not affect RecBCD-mediated DNA repair, suggesting that ExoVII, ExoI and SbcCD nucleases are not essential for the initial targeting of RecBCD to DSBs. It is possible that the DNA-blunting activity provided by ExoVII, ExoI and SbcCD is required for an exchange of RecBCD molecules on dsDNA ends during ongoing "reckless" DNA degradation.

RecF recombination pathway in Escherichia coli cells lacking RecQ, UvrD and HelD helicases.

In recBCD sbcB sbcC(D) mutants of Escherichia coli homologous recombination proceeds via RecF pathway, which is thought to require RecQ, UvrD and HelD helicases at its initial stage. It was previously suggested that depletion of all three helicases totally abolishes the RecF pathway. The present study (re)examines the roles of these helicases in transductional recombination, and in recombinational repair of UV-induced DNA damage in the RecF pathway. The study has employed the ΔrecBCD ΔsbcB sbcC201 and ΔrecBCD sbcB15 sbcC201 strains, carrying combinations of mutations in recQ, uvrD, and helD genes. We show that in ΔrecBCD ΔsbcB sbcC201 strains, recombination requires exclusively the RecQ helicase. In ΔrecBCD sbcB15 sbcC201 strains, RecQ may be partially substituted by UvrD helicase. The HelD helicase is dispensable for recombination in both backgrounds. Our results also suggest that significant portion of recombination events in the RecF pathway is independent of RecQ, UvrD and HelD. These events are initiated either by RecJ nuclease alone or by RecJ nuclease associated with an unknown helicase. Inactivation of exonuclease VII by a xseA mutation further decreases the requirement for helicase activity in the RecF pathway. We suggest that elimination of nucleases acting on 3' single-strand DNA ends reduces the necessity for helicases in initiation of recombination.

RecA protein assures fidelity of DNA repair and genome stability in Deinococcus radiodurans.

Deinococcus radiodurans is one of the most radiation-resistant organisms known. It can repair hundreds of radiation-induced double-strand DNA breaks without loss of viability. Genome reassembly in heavily irradiated D. radiodurans is considered to be an error-free process since no genome rearrangements were detected after post-irradiation repair. Here, we describe for the first time conditions that frequently cause erroneous chromosomal assemblies. Gross chromosomal rearrangements have been detected in recA mutant cells that survived exposure to 5kGy γ-radiation. The recA mutants are prone also to spontaneous DNA rearrangements during normal exponential growth. Some insertion sequences have been identified as dispersed genomic homology blocks that can mediate DNA rearrangements. Whereas the wild-type D. radiodurans appears to repair accurately its genome shattered by 5kGy γ-radiation, extremely high γ-doses, e.g., 25kGy, produce frequent genome rearrangements among survivors. Our results show that the RecA protein is quintessential for the fidelity of repair of both spontaneous and γ-radiation-induced DNA breaks and, consequently, for genome stability in D. radiodurans. The mechanisms of decreased genome stability in the absence of RecA are discussed.

Roles of ExoI and SbcCD nucleases in "reckless" DNA degradation in recA mutants of Escherichia coli.

Exponentially growing recA mutant cells of Escherichia coli display pronounced DNA degradation that starts at the sites of DNA damage and depends on RecBCD nuclease (ExoV) activity. As a consequence of this "reckless" DNA degradation, populations of recA mutants contain a large proportion of anucleate cells. We have found that both DNA degradation and anucleate-cell production are efficiently suppressed by mutations in the xonA (sbcB) and sbcD genes. The suppressive effects of these mutations were observed in normally grown, as well as in UV-irradiated, recA cells. The products of the xonA and sbcD genes are known to code for the ExoI and SbcCD nucleases, respectively. Since both xonA and sbcD mutations are required for strong suppression of DNA degradation while individual mutations have only a weak suppressive effect, we infer that ExoI and SbcCD play partially redundant roles in regulating DNA degradation in recA cells. We suggest that their roles might be in processing (blunting) DNA ends, thereby producing suitable substrates for RecBCD binding.

Bacillus subtilis strain deficient for the protein-tyrosine kinase PtkA exhibits impaired DNA replication.

Bacillus subtilis has recently come into the focus of research on bacterial protein-tyrosine phosphorylation, with several proteins kinases, phosphatases and their substrates identified in this Gram-positive model organism. B. subtilis protein-tyrosine phosphorylation system PtkA/PtpZ was previously shown to regulate the phosphorylation state of UDP-glucose dehydrogenases and single-stranded DNA-binding proteins. This promiscuity towards substrates is reminiscent of eukaryal kinases and has prompted us to investigate possible physiological effects of ptkA and ptpZ gene inactivations in this study. We were unable to identify any striking phenotypes related to control of UDP-glucose dehydrogenases, natural competence and DNA lesion repair; however, a very strong phenotype of DeltaptkA emerged with respect to DNA replication and cell cycle control, as revealed by flow cytometry and fluorescent microscopy. B. subtilis cells lacking the kinase PtkA accumulated extra chromosome equivalents, exhibited aberrant initiation mass for DNA replication and an unusually long D period.

Reassembly of shattered chromosomes in Deinococcus radiodurans.

Dehydration or desiccation is one of the most frequent and severe challenges to living cells. The bacterium Deinococcus radiodurans is the best known extremophile among the few organisms that can survive extremely high exposures to desiccation and ionizing radiation, which shatter its genome into hundreds of short DNA fragments. Remarkably, these fragments are readily reassembled into a functional 3.28-megabase genome. Here we describe the relevant two-stage DNA repair process, which involves a previously unknown molecular mechanism for fragment reassembly called 'extended synthesis-dependent strand annealing' (ESDSA), followed and completed by crossovers. At least two genome copies and random DNA breakage are requirements for effective ESDSA. In ESDSA, chromosomal fragments with overlapping homologies are used both as primers and as templates for massive synthesis of complementary single strands, as occurs in a single-round multiplex polymerase chain reaction. This synthesis depends on DNA polymerase I and incorporates more nucleotides than does normal replication in intact cells. Newly synthesized complementary single-stranded extensions become 'sticky ends' that anneal with high precision, joining together contiguous DNA fragments into long, linear, double-stranded intermediates. These intermediates require RecA-dependent crossovers to mature into circular chromosomes that comprise double-stranded patchworks of numerous DNA blocks synthesized before radiation, connected by DNA blocks synthesized after radiation.

sbcB15 And DeltasbcB mutations activate two types of recf recombination pathways in Escherichia coli.

Escherichia coli cells with mutations in recBC genes are defective for the main RecBCD pathway of recombination and have severe reductions in conjugational and transductional recombination, as well as in recombinational repair of double-stranded DNA breaks. This phenotype can be corrected by suppressor mutations in sbcB and sbcC(D) genes, which activate an alternative RecF pathway of recombination. It was previously suggested that sbcB15 and DeltasbcB mutations, both of which inactivate exonuclease I, are equally efficient in suppressing the recBC phenotype. In the present work we reexamined the effects of sbcB15 and DeltasbcB mutations on DNA repair after UV and gamma irradiation, on conjugational recombination, and on the viability of recBC (sbcC) cells. We found that the sbcB15 mutation is a stronger recBC suppressor than DeltasbcB, suggesting that some unspecified activity of the mutant SbcB15 protein may be favorable for recombination in the RecF pathway. We also showed that the xonA2 mutation, a member of another class of ExoI mutations, had the same effect on recombination as DeltasbcB, suggesting that it is an sbcB null mutation. In addition, we demonstrated that recombination in a recBC sbcB15 sbcC mutant is less affected by recF and recQ mutations than recombination in recBC DeltasbcB sbcC and recBC xonA2 sbcC strains is, indicating that SbcB15 alleviates the requirement for the RecFOR complex and RecQ helicase in recombination processes. Our results suggest that two types of sbcB-sensitive RecF pathways can be distinguished in E. coli, one that is activated by the sbcB15 mutation and one that is activated by sbcB null mutations. Possible roles of SbcB15 in recombination reactions in the RecF pathway are discussed.

The RuvABC resolvase is indispensable for recombinational repair in sbcB15 mutants of Escherichia coli.

The RuvABC proteins of Escherichia coli play an important role in the processing of Holliday junctions during homologous recombination and recombinational repair. Mutations in the ruv genes have a moderate effect on recombination and repair in wild-type strains but confer pronounced recombination deficiency and extreme sensitivity to DNA-damaging agents in a recBC sbcBC background. Genetic analysis presented in this work revealed that the (Delta)ruvABC mutation causes an identical DNA repair defect in UV-irradiated recBC sbcBC, sbcBC, and sbcB strains, indicating that the sbcB mutation alone is responsible for the extreme UV sensitivity of recBC sbcBC ruv derivatives. In experiments with gamma irradiation and in conjugational crosses, however, sbcBC (Delta)ruvABC and sbcB (Delta)ruvABC mutants displayed higher recombination proficiency than the recBC sbcBC (Delta)ruvABC strain. The frequency of conjugational recombination observed with the sbcB (Delta)ruvABC strain was quite similar to that of the (Delta)ruvABC single mutant, indicating that the sbcB mutation does not increase the requirement for RuvABC in a recombinational process starting from preexisting DNA ends. The differences between the results obtained in three experimental systems used suggest that in UV-irradiated cells, the RuvABC complex might act in an early stage of recombinational repair. The results of this work are discussed in the context of recent recombination models which propose the participation of RuvABC proteins in the processing of Holliday junctions made from stalled replication forks. We suggest that the mutant SbcB protein stabilizes these junctions and makes their processing highly dependent on RuvABC resolvase.