Mirjana Petranović

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.

RuvAB is essential for replication forks reversal in certain replication mutants.

Inactivated replication forks may be reversed by the annealing of leading‐ and lagging‐strand ends, resulting in the formation of a Holliday junction (HJ) adjacent to a DNA double‐strand end. In Escherichia coli mutants deficient for double‐strand end processing, resolution of the HJ by RuvABC leads to fork breakage, a reaction that we can directly quantify. Here we used the HJ‐specific resolvase RusA to test a putative role of the RuvAB helicase in replication fork reversal (RFR). We show that the RuvAB complex is required for the formation of a RusA substrate in the polymerase III mutants dnaEts and holD, affected for the Pol III catalytic subunit and clamp loader, and in the helicase mutant rep. This finding reveals that the recombination enzyme RuvAB targets forks in vivo and we propose that it directly converts forks into HJs. In contrast, RFR occurs in the absence of RuvAB in the dnaNts mutant, affected for the processivity clamp of Pol III, and in the priA mutant, defective for replication restart. This suggests alternative pathways of RFR.

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 ΔptkA 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.

Lethality of bypass polymerases in Escherichia coli cells with a defective clamp loader complex of DNA polymerase III.

Escherichia coli DNA polymerase III (Pol III) is one of the best studied replicative DNA polymerases. Here we report the properties of an E. coli mutant that lacks one of the subunits of the Pol III clamp loader complex, Psi (ψ), as a result of the complete inactivation of the holD gene. We show that, in this mutant, chronic induction of the SOS response in a RecFOR‐dependent way leads to lethality at high temperature. The SOS‐induced proteins that are lethal in the holD mutant are the specialized DNA polymerases Pol II and Pol IV, combined with the division inhibitor SfiA. Prevention of SOS induction or inactivation of Pol II, Pol IV and SfiA encoding genes allows growth of the holD mutant, although at a reduced rate compared to a wild‐type cell. In contrast, the SOS‐induced Pol V DNA polymerase does not participate to the lethality of the holD mutant. We conclude that: (i) Psi is essential for efficient replication of the E. coli chromosome; (ii) SOS‐induction of specialized DNA polymerases can be lethal in cells in which the replicative polymerase is defective, and (iii) specialized DNA polymerases differ in respect to their access to inactivated replication forks.

Genetic evidence that the elevated levels of Escherichia coli helicase II antagonize recombinational DNA repair

Some phages survive irradiation much better upon multiple than upon single infection, a phenomenon known as multiplicity reactivation (MR). Long ago MR of UV-irradiated lambda red phage in E. coli cells was shown to be a manifestation of recA-dependent recombinational DNA repair. We used this experimental model to assess the influence of helicase II on the type of recombinational repair responsible for MR. Since helicase II is encoded by the SOS-inducible uvrD gene, SOS-inducing treatments such as irradiating recA(+) or heating recA441 cells were used. We found: i) that MR was abolished by the SOS-inducing treatments; ii) that in uvrD background these treatments did not affect MR; and iii) that the presence of a high-copy plasmid vector carrying the uvrD(+) allele together with its natural promoter region was sufficient to block MR. From these results we infer that helicase II is able to antagonize the type of recA-dependent recombinational repair acting on multiple copies of UV-damaged lambda DNA and that its anti-recombinogenic activity is operative at elevated levels only.

sbcB15 and ΔsbcB 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.

Different reparability of the chromosomal and cytoplasmic deoxyribonucleic acid in Escherichia coli damaged by gamma and ultraviolet irradiation.

We have assessed the relative efficiencies by which chromosomal and extrachromosomal DNAs are repaired in irradiated bacteria. Repair-proficient Escherichia coli C600 cells lysogenic for, or infected with, the thermoinducible phage λcI857 ind were exposed to γ or ultraviolet radiation and then tested for colony- and plaque-forming ability. The results show that the bacterial cell is about 5 times more sensitive to γ rays and about 1.5 times more sensitive to ultraviolet light, if compared to either (1) the prophage that is irradiated in the bacterial chromosome and, on heat induction, repaired in the cytoplasm, or (2) the infecting phage that is irradiated and repaired in the cytoplasm. Since the bacterial DNA is about 80 times larger than the phage DNA, it is inferred that repair processes operating along the chromosomal DNA are one order of magnitude more efficient than those operating along the extrachromosomal DNA. This conclusion is reinforced by the fact that the absence of repair in the system Esch...

Exonuclease Requirements for Recombination of λ-Phage in recD Mutants of Escherichia coli

Recombination of λ red gam phage in recD mutants is unaffected by inactivation of RecJ exonuclease. Since nucleases play redundant roles in E. coli, we inactivated several exonucleases in a recD mutant and discovered that 5′–3′ exonuclease activity of RecJ and exonuclease VII is essential for λ-recombination, whereas exonucleases of 3′–5′ polarity are dispensable. The implications of the presented data on current models for recombination initiation in E. coli are discussed.

W reactivation is inefficient in repair of the bacterial chromosome.

SummaryUV-inducible “SOS” processes associated with W reactivation of phage lambda were studied for their effect on repair of lambda prophage integrated in the bacterial chromosome. For this purpose, lambda cI857 ind red-lysogens were used. These lysogens, although non-inducible by UV light, can be induced by raising the temperature from 30° to 42°. If the W reactivation processes are involved in repair of the bacterial DNA, when the lysogens are incubated at 30° after UV exposure W reactivation should be fully expressed and should also exert an effect on the bacterial chromosome and the prophage inside it. When heat-induction is delayed until the time at which W reactivation reaches its maximum, a considerable increase in phage survival might then be expected. The results presented in this report show, however, that the delayed induction had only a small effect on the survival of prophage in the wild-type strain (possibly attributable to excision repair) and no detectable effect on prophage in a uvrA strain. From these results we conclude that W reactivation is largely irrelevant to the repair of UV-damaged bacterial DNA.