Repair Of Daughter-strand Gaps Research Articles

Recombinational repair in the absence of holliday junction resolvases in E. coli.

Cells possess two major DNA damage tolerance pathways that allow them to duplicate their genomes despite the presence of replication blocking lesions: translesion synthesis (TLS) and daughter strand gap repair (DSGR). The TLS pathway involves specialized DNA polymerases that are able to synthesize past DNA lesions while DSGR relies on Recombinational Repair (RR). At least two mechanisms are associated with RR: Homologous Recombination (HR) and RecA Mediated Excision Repair (RAMER). While HR and RAMER both depend on RecFOR and RecA, only the HR mechanism should involve Holliday Junctions (HJs) resolvase reactions. In this study we investigated the role of HJ resolvases, RuvC, TopIII and RusA on the balance between RAMER and HR in E. coli MG1655 derivatives. Using UV survival measurements, we first clearly establish that, in this genetic background, topB and ruvC define two distinct pathways of HJ resolution. We observed that a recA mutant is much more sensitive to UV than the ruvC topB double mutant which is deficient in HR because of its failure to resolve HJs. This difference is independent of RAMER, the SOS system, RusA, and the three TLS DNA polymerases, and may be accounted for by Double Strand Break repair mechanisms such as Synthesis Dependent Strand Annealing, Single Strand Annealing, or Break Induced Replication, which are independent of HJ resolvases. We then used a plasmid-based assay, in which RR is triggered by a single blocking lesion present on a plasmid molecule, to establish that while HR requires topB, ruvC or rusA, RAMER is independent of these genes and, as expected, requires a functional UvrABC excinuclease. Surprisingly, analysis of the RR events in a strain devoid of HJ resolvases reveals that the UvrABC dependent repair of the single lesion present on the plasmid molecule can generate an excision track potentially extending to dozens of nucleotides.

Replication Forks Stalled at Ultraviolet Lesions Are Rescued via RecA and RuvABC Protein-catalyzed Disintegration in Escherichia coli

Ultraviolet (UV) irradiation is not known to induce chromosomal fragmentation in sublethal doses, and yet UV irradiation causes genetic instability and cancer, suggesting that chromosomes are fragmented. Here we show that UV irradiation induces fragmentation in sublethal doses, but the broken chromosomes are repaired or degraded by RecBCD; therefore, to observe full fragmentation, RecBCD enzyme needs to be inactivated. Using quantitative pulsed field gel electrophoresis and sensitive DNA synthesis measurements, we investigated the mechanisms of UV radiation-induced chromosomal fragmentation in recBC mutants, comparing five existing models of DNA damage-induced fragmentation. We found that fragmentation depends on active DNA synthesis before, but not after, UV irradiation. At low UV irradiation doses, fragmentation does not need excision repair or daughter strand gap repair. Fragmentation absolutely depends on both RecA-catalyzed homologous strand exchange and RuvABC-catalyzed Holliday junction resolution. Thus, chromosomes fragment when replication forks stall at UV lesions and regress, generating Holliday junctions. Remarkably, cells specifically utilize fork breakage to rescue stalled replication and avoid lethality.

Role of homologous recombination in DNA interstrand crosslink repair

Homologous recombination repair (HRR) encompasses mechanisms that employ homologous DNA sequences as templates for repair or tolerance of a wide range of DNA lesions that inhibit DNA replication in S phase. Arguably the most imposing of these DNA lesions is that of the interstrand crosslink (ICL), consisting of a covalently attached chemical bridge between opposing DNA strands. ICL repair requires the coordinated activities of HRR and a number of proteins from other DNA repair and damage response systems, including nucleotide excision repair, base excision repair, mismatch repair, and translesion DNA synthesis (TLS). Interestingly, different organisms favor alternative methods of HRR in the ICL repair process. E. coli perform ICL repair using a homology-driven damage bypass mechanism analogous to daughter strand gap repair. Eukaryotes from yeast to humans initiate ICL repair primarily during DNA replication, relying on HRR activity to restart broken replication forks associated with double-strand break intermediates induced by nucleolytic activities of other excision repair factors. Higher eukaryotes also employ several additional factors, including members of the Fanconi anemia damage-response network, which further promote replication-associated ICL repair through the activation and coordination of various DNA excision repair, TLS, and HRR proteins. This review focuses on the proteins and general mechanisms of HRR associated with ICL repair in different model organisms.

Escherichia coli responses to a single DNA adduct.

To study the mechanisms by which Escherichia coli modulates the genotoxic effects of DNA damage, a novel system has been developed which permits quantitative measurements of various E. coli pathways involved in mutagenesis and DNA repair. Events measured include fidelity and efficiency of translesion DNA synthesis, excision repair, and recombination repair. Our strategy involves heteroduplex plasmid DNA bearing a single site-specific DNA adduct and several mismatched regions. The plasmid replicates in a mismatch repair-deficient host with the mismatches serving as strand-specific markers. Analysis of progeny plasmid DNA for linkage of the strand-specific markers identifies the pathway from which the plasmid is derived. Using this approach, a single 1, N(6)-ethenodeoxyadenosine adduct was shown to be repaired inefficiently by excision repair, to inhibit DNA synthesis by approximately 80 to 90%, and to direct the incorporation of correct dTMP opposite this adduct. This approach is especially useful in analyzing the damage avoidance-tolerance mechanisms. Our results also show that (i) progeny derived from the damage avoidance-tolerance pathway(s) accounts for more than 15% of all progeny; (ii) this pathway(s) requires functional recA, recF, recO, and recR genes, suggesting the mechanism to be daughter strand gap repair; (iii) the ruvABC genes or the recG gene is also required; and (iv) the RecG pathway appears to be more active than the RuvABC pathway. Based on these results, the mechanism of the damage avoidance-tolerance pathway is discussed.

ATP Hydrolysis and DNA Binding by the Escherichia coli RecF Protein

The Escherichia coli RecF protein possesses a weak ATP hydrolytic activity. ATP hydrolysis leads to RecF dissociation from double-stranded (ds)DNA. The RecF protein is subject to precipitation and an accompanying inactivation in vitro when not bound to DNA. A mutant RecF protein that can bind but cannot hydrolyze ATP (RecF K36R) does not readily dissociate from dsDNA in the presence of ATP. This is in contrast to the limited dsDNA binding observed for wild-type RecF protein in the presence of ATP but is similar to dsDNA binding by wild-type RecF binding in the presence of the nonhydrolyzable ATP analog, adenosine 5'-O-(3-thio)triphosphate (ATPgammaS). In addition, wild-type RecF protein binds tightly to dsDNA in the presence of ATP at low pH where its ATPase activity is blocked. A transfer of RecF protein from labeled to unlabeled dsDNA is observed in the presence of ATP but not ATPgammaS. The transfer is slowed considerably when the RecR protein is also present. In competition experiments, RecF protein appears to bind at random locations on dsDNA and exhibits no special affinity for single strand/double strand junctions when bound to gapped DNA. Possible roles for the ATPase activity of RecF in the regulation of recombinational DNA repair are discussed.

Escherichia coli strains lacking protein HU are UV sensitive due to a role for HU in homologous recombination.

hupA and hupB encode the alpha and beta subunits of the Escherichia coli histone-like protein HU. Here we show that E. coli hup mutants are sensitive to UV in the rec+ sbc+, recBC sbcA, recBC sbcBC, umuDC, recF, and recD backgrounds. However, hupAB mutations do not enhance the UV sensitivity of resolvase-deficient recG ruvA strains. hupAB uvrA and hupAB recG strains are supersensitive to UV. hup mutations enhance the UV sensitivity of ruvA strains to a much lesser extent but enhance that of rus-1 ruvA strains to the same extent as for rus+ ruv+ strains. Our results suggest that HU plays a role in recombinational DNA repair that is not specifically limited to double-strand break repair or daughter strand gap repair; the lack of HU affects the RecG RusA and RuvABC pathways for Holliday junction processing equally if the two pathways are equally active in recombinational repair; the function of HU is not in the substrate processing step or in the RecFOR-directed synapsis action during recombinational repair. Furthermore, the UV sensitivity of hup mutants cannot be suppressed by overexpression of wild-type or mutant gyrB, which confers novobiocin resistance, or by different concentrations of a gyrase inhibitor that can increase or decrease the supercoiling of chromosomal DNA.

Evolution of the SNF2 family of proteins: subfamilies with distinct sequences and functions.

The SNF2 family of proteins includes representatives from a variety of species with roles in cellular processes such as transcriptional regulation (e.g. MOT1, SNF2 and BRM), maintenance of chromosome stability during mitosis (e.g. lodestar) and various aspects of processing of DNA damage, including nucleotide excision repair (e.g. RAD16 and ERCC6), recombinational pathways (e.g. RAD54) and post-replication daughter strand gap repair (e.g. RAD5). This family also includes many proteins with no known function. To better characterize this family of proteins we have used molecular phylogenetic techniques to infer evolutionary relationships among the family members. We have divided the SNF2 family into multiple subfamilies, each of which represents what we propose to be a functionally and evolutionarily distinct group. We have then used the subfamily structure to predict the functions of some of the uncharacterized proteins in the SNF2 family. We discuss possible implications of this evolutionary analysis on the general properties and evolution of the SNF2 family.

Involvement of RecF pathway recombination genes in postreplication repair in UV-irradiated Escherichia coli cells

Mutations affecting the RecF pathway of recombination (recF, recG, recJ, recN, recO, recQ, recR, ruvA, ruvC) were systematically introduced into two sets of strains: (a) uvrA and uvrA recA2020, (b) uvrA recBC sbcBC and uvrA recBC sbcBC recA2020. We examined: (i) the effect of these mutations on the repair of DNA daughter-strand gaps which are produced in the nascent DNA synthesized after UV irradiation, and (ii) the ability of recA2020 (a suppressor for the recF mutation) to suppress the UV radiation sensitivity caused by these mutations. In the uvrA cells, mutations in recF, recR or recO genes produced a major deficiency in the repair of daughter-strand gaps, whereas mutations in recJ, recG, recN, recQ, ruvA or ruvC genes had no effect on the repair of daughter-strand gaps. In both uvrA and uvrA recBC sbcBC backgrounds, the UV radiation sensitivity caused by recF, recG, recR, recO, ruvA, or ruvC mutation was partially suppressed by recA2020, whereas the UV radiation sensitivity caused by recJ, recN, or recQ mutations was not suppressed by recA2020. Partial suppression of the UV sensitivity of recG, ruvA and ruvC mutants was not observed with other suppressors for recF, i.e., recA441, recA720 and recA730. Taken together, these results further support the notion that the recF, recR and recO gene products (abbreviated as RecFOR) function at the same step in recombination repair, possible as a complex. It also suggests that this putative RecFOR) complex does not contain proteins encoded by other genes involved in the RecF pathway of recombination.

Hypothesis. RuvA, RuvB and RuvC proteins: Cleaning‐up after recombinational repairs in E. coli

After the completion of RecA protein-mediated recombinational repair of daughter-strand gaps in E. coli, participating chromosomes are held together by Holliday junctions. Until recently, it was not known how the cell disengages the connected chromosomes. Accumulating genetic data suggested that the product of the ruv locus participates in recombinational repair and acts after the formation of Holliday junctions. Molecular characterization of the locus revealed that there are three genes--ruvA, ruvB and ruvC; mutations in any one of the genes confer the same phenotype. Recently, the RuvC protein was found to be a Holliday junction resolvase. At first glance, the resolving activity of RuvC alone would appear to be sufficient for the separation of recombining chromosomes. However, in vitro studies show that the filament of RecA protein is unable to dissociate from the products of the recombination reaction. Thus, in vivo, even if the Holliday junctions are resolved by RuvC, RecA filament must be holding two DNA duplexes together. New findings about enzymatic activities of RuvA and RuvB proteins foster the hope that the machinery for removing the RecA filament from DNA has been found.

RecA-dependent DNA repair processes.

UV-radiation-induced lesions in DNA result in the formation of: (1) excision gaps (i.e. a lesion is excised, leaving a gap), (2) daughter-strand gaps (i.e. a lesion can be skipped during replication, leaving a gap), and (3) double-strand breaks (i.e. the DNA strand opposite a gap can be cut). In Escherichia coli, the recA gene product is involved in repairs of all three types of lesions--repair of daughter-strand gaps (2) and double-strand breaks (3) constitutes post-replication repair. The evidence suggests, furthermore, that recA-dependent repair of excision gaps (1) produced in DNA replicated prior to UV irradiation (pre-replication repair) appears to occur by similar mechanisms.

Temperature dependent survival of UV-irradiated Escherichia coli K12

We have found that several excision deficient derivatives of Escherichia coli K12 survive better after UV irradiation if incubated at 42 degrees C than if incubated at 30 degrees C. The highest survival was observed when incubation at 42 degrees C followed UV irradiation and was maintained for at least 16 h. Our results indicate that this temperature dependent resistance (TDR) requires a functional recA gene, but not uvrA, uvrB, recF, or recB genes, or the recA441 (tif-1) mutation which allows thermoinduction of the recA-lexA regulon. Our data are consistent with the idea that the increase in survival observed at 42 degrees C reflects enhanced daughter-strand gap repair by DNA strand exchange. Although the conditions used to elicit TDR can induce heat shock proteins and thermotolerance in E. coli, the relationship between the two responses remains to be elucidated.

Different effects of recJ and recN mutations on the postreplication repair of UV-damaged DNA in Escherichia coli K-12.

Two mutations known to affect recombination in a recB recC sbsBC strain, recJ284::Tn10 and recN262, were examined for their effects on the postreplication repair of UV-damaged DNA. The recJ mutation did not affect the UV radiation sensitivity of uvrB and uvrB recF cells, but it increased the sensitivity of uvrB recN (approximately 3-fold) and uvrB recB (approximately 8-fold) cells. On the other hand, the recN mutation did not affect the UV sensitivity of uvrB recB cells, but it increased the sensitivity of uvrB (approximately 1.5-fold) and uvrB recF (approximately 4-fold) cells. DNA repair studies indicated that the recN mutation produced a partial deficiency in the postreplication repair of DNA double-strand breaks that arise from unrepaired daughter strand gaps, while the recJ mutation produced a deficiency in the repair of daughter strand gaps in uvrB recB cells (but not in uvrB cells) and a deficiency in the repair of both daughter strand gaps and double-strand breaks in uvrA recB recC shcBC cells. Together, these results indicate that the recJ and recN genes are involved in different aspects of postreplication repair.

The unique sensitivity of Walker rat tumour cells to difunctional agents is associated with a failure to recover from inhibiton of DNA synthesis and increased chromosome damage

The rate and modle of DNA synthesis was examined by thymidine uptake and by flow cytometry in Walker tumour cells highly sensitive to difunctional agents (WS), and in a derived subline of resistant cells (WR) (Rawlings and Roberts, 1986), following their treatment with sulphur mustard. Both cell lines exhibited the same dose-dependent and progressive depression in rate of DNA synthesis for up h after treatment. Thereafter the depression in rate of synthesis was partially reserved in the WR cells but DNA synthesis continued to decrease in the WS cells resulting in their slower transit through the S phase and a persistent block in the G 2/M phase of the cell cycle. Sensitive cells which finally escaped the block in G 2 carried more chromosome aberrations than the corresponding resistant cells. Neither cell line was defective in daughter strand-gap repair. In the sensitivity to difunctional but not to monofunctional compounds, their failure to recover from the early depression of DNA synthesis, their apparent lack of a defect in excision repair and their sensitivity to chromosome aberration induction, the Walker cell phenmotype closely resembles that of the human Fanconi's anaemia cell.

A mechanism for rich-medium inhibition of the repair of daughter-strand gaps in the deoxyribonucleic acid of UV-irradiated Escherichia coli K12 uvrA

Ultraviolet-irradiated Escherichia coli K12 uvrA(B,C) cells show higher survival if plated on minimal growth medium (MM) rather than on rich growth medium (RM). This phenomenon has been referred to as ‘minimal medium recovery’ (MMR). UV-irradiated (4 J/m 2) uvrA cells showed a similar rate of protein synthesis, whether incubated in MM or RM, however, they showed a severe depression in DNA synthesis when incubated in MM that lasted for about 30 min, and the normal rate of DNA synthesis was not reestablished until about 60 min after irradiation. When a sample of these same cells was switched to RM immediately after UV-irradiation, there was only a slight slowing of DNA synthesis, and the normal rate of synthesis was reestablished by 60 min. An additional mmrA mutation or growth retardation by valine blocked both this extra DNA synthesis in RM, and the inhibitory effect of RM on survival. These findings suggest that the absence of a marked delay in DNA synthesis observed in RM may be responsible for the inhibitory effect of RM on the survival of UV-irradiated excision-deficient cells. Two hypotheses, which are not mutually exclusive, are propose and supported by data to explain why a fast rate of DNA synthesis after UV-irradiation partially inhibits postreplication repair and enhances cell lethality.

Mechanisms for recF-dependent and recB-dependent pathways of postreplication repair in UV-irradiated Escherichia coli uvrB.

The molecular mechanisms for the recF-dependent and recB-dependent pathways of postreplication repair were studied by sedimentation analysis of DNA from UV-irradiated Escherichia coli cells. When the ability to repair DNA daughter strand gaps was compared, uvrB recF cells showed a gross deficiency, whereas uvrB recB cells showed only a small deficiency. Nevertheless, the uvrB recF cells were able to perform some limited repair of daughter strand gaps compared with a "repairless" uvrB recA strain. The introduction of a recB mutation into the uvrB recF strain greatly increased its UV radiation sensitivity, yet decreased only slightly its ability to repair daughter strand gaps. Kinetic studies of DNA repair with alkaline and neutral sucrose gradients indicated that the accumulation of unrepaired daughter strand gaps led to the formation of low-molecular-weight DNA duplexes (i.e., DNA double-strand breaks were formed). The uvrB recF cells were able to regenerate high-molecular-weight DNA from these low-molecular-weight DNA duplexes, whereas the uvrB recF recB and uvrB recA cells were not. A model for the recB-dependent pathway of postreplication repair is presented.

Repair of daughter strand gaps in nascent DNA from mouse epidermal cells treated with dihydrodiol epoxide derivatives of benzo[ a]pyrene

Alkaline sucrose gradient analysis of [ methyl- 3H]thymidine-pulse-labeled DNA was used to study the effect of (±)-7 β,8 a-dihydroxy-9 α,10 α-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene (benzo[ a]pyrene-diol epoxide I), a potent mutagen and carcinogen, and (±)-7 β,8 α-dihydroxy-9 β,10 β-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene (benzo[ a]pyrene-diol epoxide II), a weaker mutagen and carcinogen, on the size of newly synthesized DNA in primary cultures of mouse epidermal cells. Both isomers caused a dose-dependent decrease in the size of newly synthesized DNA and in the rate of [ methyl- 3H]thymidine incorporation into DNA. When the pulse time was increased in the treated cells so that the amount of [ methyl- 3H]thymidine incorporation was equal to the control, newly synthesized DNA from exposed cells was still considerably smaller than DNA from control cells. The low molecular weight of the nascent DNA from treated cells was consistent with, but not indicative of, the presence of gaps in the nascent DNA from the treated cells. Evidence of gapped DNA synthesis was obtained by treatment of extracted DNA with a single-strand specific endonuclease from Neurospora crassa. The endonuclease treatment did not significantly alter the profile of [ methyl- 3H]thymidine prelabeled DNA from benzo[ a]pyrene-diol epoxide-treated cultures but did introduce double-strand breaks in pulse-labeled DNA from treated cultures. The numbers of [ 14C]benzo[ a]pyrene-diol epoxide I or [ 3H]benzo[ a]pyrene-diol epoxide II-DNA-bound adducts and daughter strand gaps were compared at several dose levels. Treatment with either isomer yielded one gap in the nascent DNA DNA-bound adduct. Pulse-chase experiments showed that gaps in the nascent DNA were closed with time.

Action of Nitrofurans on E. Coli: Mutation and induction and repair of daughter-strand gaps in DNA

The antibacterial and mutagenic potency of 9 nitrofurans in “treat and plate” experiments varied over almost 5 orders of magnitude. The relative toxicities were as follows: FANFT > AF2 > ANFT > furazolidone > furagin ⪢ nitrofurantoin > nitrofurazone ⪢ methylnitrofuroate ⪢ nitrofuroic acid. In general, mutagenic activity paralleled toxicity. The compounds at concentrations corresponding to their LD 50's, induced mutations at frequencies which ranged from 2.5 10 6 survivors for FANFT to 130 10 6 survivors for furagin (NF416). The observed differences in antibacterial and mutagenic activity are unlikely to be due to lack of activation of the weaker agents since the two most potent agents were reduced somewhat more slowly than many of the less active agents. The relative sensitivities to the antibacterial effects of AF2 of strains WP2, WP2 uvrA, CM561 ( lexA) and CM571 ( recA) were 1 :1.6:3:7 and to nitrofurazone 1 : 1 : 25 : 50. The uvrA strain was 6–7-fold more mutable with both these agents than was WP2. No increase over the spontaneous mutation frequency was observed when recA or lexA strains were exposed to either AF2 or nitrofurazone in these experiments. When wild-type of uvrA bacteria containing nitrofuran-induced lesions replicated their DNA in drug-free medium in the presence of [ 3H]thymidine for 5 min, the label was found in low molecular weight DNA indicating that daughter-strand gaps were formed. During subsequent incubation in nonradioactive medium the molecular weight of the DNA increased to the control value. A recA strain (which was very sensitive to the lethal effects of AF2 and nitrofurazone) lacked the ability to repair daughter-strand gaps caused by nitrofuran-induced lesions.