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1.
Biochimie ; 83(11-12): 1041-7, 2001.
Article in English | MEDLINE | ID: mdl-11879732

ABSTRACT

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.


Subject(s)
Adenosine Triphosphatases/metabolism , Bacterial Proteins/genetics , Bacteriophage lambda/radiation effects , DNA Helicases/metabolism , DNA Repair/drug effects , Escherichia coli Proteins , SOS Response, Genetics/genetics , Transcription Factors , Ultraviolet Rays , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/pharmacology , Bacterial Proteins/drug effects , Bacteriophage lambda/drug effects , Bacteriophage lambda/genetics , DNA Helicases/genetics , DNA Helicases/pharmacology , DNA Repair/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression Regulation , Rec A Recombinases/pharmacology , Rec A Recombinases/radiation effects , Recombination, Genetic/drug effects , Ultraviolet Rays/adverse effects
2.
Res Microbiol ; 152(10): 873-81, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11766962

ABSTRACT

Earlier studies have revealed a radiation-induced process leading to the loss of lambda prophage recombinogenicity. The process takes place in UV-irradiated Escherichia coli cells, and renders the prophage incapable of site-specific recombination with the host chromosome, and of general recombination with an infecting homologous phage. It was found that the inhibition of prophage recombinogenicity depends on functional RecBCD enzyme of E. coli. In this work, the role of ruvABC and recG genes in the inhibitory process was assessed. The products of these genes are known to act at the last step of homologous recombination and recombinational DNA repair by catalyzing the resolution of recombination intermediates (the Holliday junctions). Irradiated prophage retained its ability to recombine in ruvA, ruvB, ruvC, and recG mutants. These results suggest that in addition to RecBCD enzyme, RuvABC and RecG proteins are also involved in the inhibition of prophage recombinogenicity. We infer that RuvABC and RecG act in this process before RecBCD, probably by processing the Holliday junctions formed upon replication arrest, and thereby providing double-stranded DNA breaks as substrate for RecBCD-mediated recombinational repair of UV-damaged bacterial chromosome.


Subject(s)
Bacteriophage lambda/physiology , DNA Helicases , Escherichia coli Proteins , Escherichia coli/virology , Bacterial Proteins/genetics , Bacteriophage lambda/genetics , DNA Repair , DNA, Bacterial/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/radiation effects , Endodeoxyribonucleases/genetics , Escherichia coli/genetics , Escherichia coli/radiation effects , Exodeoxyribonuclease V , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/radiation effects , Mutation , Recombination, Genetic , Ultraviolet Rays , Virus Activation
3.
Res Microbiol ; 151(9): 727-38, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11130863

ABSTRACT

RecBCD enzyme is involved in the radiation-induced process known as prophage inactivation. The process leads to the inability of lambda prophage to excise itself from the Escherichia coli chromosome via site-specific recombination. In this work we sought to further characterize the role of RecBCD enzyme in this process. In addition, we examined the ability of irradiated prophage to recombine with the infecting homologous phage. We used several E. coli mutants differentially altered in RecBCD's activities. The results showed that in the mutants carrying either recB2109 or recD1903, which do not exhibit significant nuclease activities, the prophage progressively loses its capacity for both site-specific and general recombination. In the recB268 null mutant, however, prophage recombinogenicity remained fully preserved. We also showed that the prophage unable to recombine retained its ability to complement the mutant infecting phage and that the recombination frequencies in phage x phage crosses were not affected by postirradiation incubation. Our results suggest that the helicase activity of RecBCD is responsible for the progressive loss of prophage recombinogenicity. This loss is most probably a consequence of the unsuccessful RecBCD-dependent recombinational repair of double-stranded breaks in the cell chromosome, during which some structures unsuitable for further recombination reactions may be produced.


Subject(s)
Bacteriophage lambda/genetics , Escherichia coli/virology , Exodeoxyribonucleases/metabolism , Recombination, Genetic , Ultraviolet Rays , Bacteriophage lambda/pathogenicity , Bacteriophage lambda/physiology , DNA Repair , Escherichia coli/enzymology , Escherichia coli/growth & development , Escherichia coli/radiation effects , Exodeoxyribonuclease V , Virus Activation
4.
Neoplasma ; 47(6): 375-81, 2000.
Article in English | MEDLINE | ID: mdl-11263862

ABSTRACT

The efficiency of Xenopus laevis egg extract to repair T:G and A:C mismatched base pairs in unmethylated, hemimethylated and fullymethylated heteroduplexes was investigated. Filamentous phage M13mp18 and its derivative M13mp18/MP-1 (C changed to T inside the sequence dCC*C GGG, at the position 6248) were used for heteroduplexes construction. The three origins of mismatched base-pairs in the eukaryotic DNA are mimicked by in vitro methylation: hemimethylated DNA (me-/me+) for replication errors; unmethylated (me-/me-) and fully methylated DNA (me+/me+) for recombination heteroduplexes, and fullymethylated also for locally, spontaneously deaminated 5-methylcytosine (5meC) to T, generating the exclusively T:G mismatch. The methylations were in CpG dinucleotides, mostly characteristic ofeukaryotic cells [5, 24]. In vitro methylation was done by HpaII methylase which methylate central C of dCCGG sequence in the manner of eukaryotic methylation. The position of mismatched bases was chosen so that correction of mismatched bases in any strand would create the sequence for one of the "diagnostic" restriction endonucleases, either BstNI or MspI. Correction efficiency was about 10(8) repair events per egg equivalent. Correction in favor of C:G base pair restoration occurred regardless of the T:G or C:A mispairs, with almost equal efficiency. Repair of T:G to T:A was up to 10 times less efficient comparing to C:G, and repair of C:A to T:A was in our experimental system undetectable. No significant difference in repair efficiency of mismatched bases situated in unmethylated, hemimethylated or fullymethylated heteroduplexes indicate methylation-independent repair of mismatched bases in X. laevis oocite extracts.


Subject(s)
Base Pair Mismatch , DNA Methylation , DNA Repair , Ovum/metabolism , Animals , CpG Islands , DNA-Cytosine Methylases/metabolism , Heteroduplex Analysis , Plasmids/metabolism , Xenopus
5.
J Bacteriol ; 181(19): 6179-83, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10498734

ABSTRACT

The RuvC protein is important for DNA recombination and repair in Escherichia coli. The present work shows that a ruvC null mutation introduced into a recBC sbcBC background causes severe defects in chromosome segregation and cell division. Both defects were found to result from abortive recombination initiated by the RecA protein.


Subject(s)
Bacterial Proteins/genetics , Chromosomes, Bacterial/ultrastructure , Endodeoxyribonucleases/genetics , Escherichia coli Proteins , Escherichia coli/genetics , Exodeoxyribonucleases/genetics , Chromosome Segregation , DNA Replication/genetics , DNA, Bacterial/biosynthesis , Exodeoxyribonuclease V , Recombination, Genetic/genetics
6.
EMBO J ; 13(11): 2725-34, 1994 Jun 01.
Article in English | MEDLINE | ID: mdl-8013470

ABSTRACT

Nearly precise excision of a transposon related to Tn10 from an Escherichia coli plasmid was used as a model to study illegitimate DNA recombination between short direct repeats. The excision was stimulated 100-1000 times by induction of plasmid single-stranded DNA synthesis and did not involve transfer of DNA from the parental to the progeny molecule. We conclude that it occurred by copy-choice DNA recombination, and propose that other events of recombination between short direct repeats might be a result of the same process.


Subject(s)
DNA Transposable Elements/genetics , Multigene Family/genetics , Plasmids/metabolism , Recombination, Genetic/genetics , Repetitive Sequences, Nucleic Acid , Base Sequence , DNA Replication/genetics , DNA, Recombinant/metabolism , DNA, Single-Stranded/biosynthesis , Escherichia coli/genetics , Escherichia coli/growth & development , Inoviridae/physiology , Models, Genetic , Molecular Sequence Data , Nucleic Acid Conformation , Plasmids/genetics , Viral Proteins/genetics , Viral Proteins/metabolism
7.
Gene ; 135(1-2): 161-6, 1993 Dec 15.
Article in English | MEDLINE | ID: mdl-8276254

ABSTRACT

Illegitimate recombination, which is one of the major causes of genome rearrangements, can occur in a number of ways. These might involve enzymes which cut and join DNA or enzymes which replicate DNA, as illustrated by two examples: (i) formation of deletions at the replication origin (ori) of an Escherichia coli bacteriophage, M13; and (ii) excision of E. coli transposon Tn10. It is proposed that a common theme to various ways by which illegitimate recombination can occur might be the capacity to create ends in the DNA molecule and to make the ends meet.


Subject(s)
Recombination, Genetic , Bacteriophage M13/genetics , DNA Transposable Elements , Escherichia coli/genetics
8.
Biochimie ; 73(4): 515-7, 1991 Apr.
Article in English | MEDLINE | ID: mdl-1911952

ABSTRACT

The mechanism of DNA replication in ultraviolet (UV)-irradiated Escherichia coli is proposed. Immediately after UV exposure, the replisome aided by single-strand DNA-binding protein (SSB) can proceed past UV-induced pyrimidine dimers without insertion of nucleotides. Polymerisation eventually resumes somewhere downstream of the dimer sites. Due to the limited supply of SSB, only a few dimers can be bypassed in this way. Nevertheless, this early DNA synthesis is of great biological importance because it generates single-stranded DNA regions. Single-stranded DNA can bind and activate RecA protein, thus leading to induction of the SOS response. During the SOS response, the cellular level of RecA protein increases dramatically. Due to the simultaneous increase in the concentration of ATP, RecA protein achieves the high-affinity state for single-stranded DNA. Therefore it is able to displace DNA-bound SSB. The cycling of SSB on and off DNA enables the replisome to bypass a large number of dimers at late post-UV times. During this late replication, the stoichiometric amounts of RecA protein needed for recombination are involved in the process of postreplication repair.


Subject(s)
DNA Replication/radiation effects , DNA, Bacterial/biosynthesis , DNA, Single-Stranded/biosynthesis , Rec A Recombinases/genetics , DNA, Bacterial/radiation effects , DNA, Single-Stranded/radiation effects , Rec A Recombinases/biosynthesis , Rec A Recombinases/radiation effects , Ultraviolet Rays
9.
Nucleic Acids Res ; 18(8): 2159-62, 1990 Apr 25.
Article in English | MEDLINE | ID: mdl-2159641

ABSTRACT

DNAs from phage mutants M13mp18 and M13mp18/MP-1 were used to construct two closed circular heteroduplexes. One of them carried the sequence 5'-CCTGGG-3' 3'-GGGCCC-5' with a T.G mismatch at the position 6248. The other carried the sequence 5'-CCCGGG-3' 3'-GGACCC-5' with a C.A mismatch at the same position. Heteroduplexes were exposed to 7 restriction endonucleases having recognition sites within the sequence 5'-CCCGGG-3' 3'-GGGCCC-5' and to 1 restriction endonuclease having a recognition site within the sequence 5'-CCTGGG-3' 3'-GGACCC-5'. All tested enzymes cleaved at least one mismatch-containing sequence although with reduced efficiency. Smal and Xmal tolerated both mismatch-containing sequences. Aval, Hpall, Mspl, Ncil and Nsplll were able to tolerate only the T.G containing sequence, while BstNl was able to tolerate only the C.A containing sequence. It is inferred that the tolerance displayed by Smal and Xmal depends on the presence of either the original purines or the original pyrimidines in mismatches of both the T.G and C.A type and that all other tested enzymes require the presence of the original purines in mismaches of both types.


Subject(s)
Bacteriophages/genetics , DNA Restriction Enzymes/metabolism , DNA, Viral/metabolism , Nucleic Acid Heteroduplexes/metabolism , Base Composition , Base Sequence , Mutation , Purine Nucleotides , Pyrimidine Nucleotides
10.
Mutat Res ; 184(1): 1-6, 1987 Jul.
Article in English | MEDLINE | ID: mdl-3299072

ABSTRACT

By making use of the temperature-sensitive mutant recB270, we showed that the RecBCD enzyme is needed for repair between 1 and 4 h after UV exposure. recB-dependent prophage inactivation (Petranovic et al. (1984), Mol. Gen. Genet., 196, 167-169) takes place in all dying cells during the same period of time. The kinetics of decrease in the yield of recombinants in phage-propage crosses resemble those of prophage inactivation in UV-irradiated bacteria. This indicates that recombination processes (including site-specific recombination required for prophage excision) are blocked in cells destined to die. On the basis of our results, we suggest that a large fraction of damaged cells is rescued by the RecA-RecBCD recombination pathway. If repair is unsuccessful, RecA-RecBCD recombination intermediates persist in the irradiated cells leading to prophage inactivation.


Subject(s)
Coliphages/radiation effects , DNA Repair , Escherichia coli Proteins , Escherichia coli/radiation effects , Exodeoxyribonucleases/metabolism , Ultraviolet Rays , Coliphages/enzymology , Coliphages/genetics , Escherichia coli/enzymology , Escherichia coli/genetics , Exodeoxyribonuclease V , Kinetics
11.
Article in English | MEDLINE | ID: mdl-6365817

ABSTRACT

DNA degradation in Escherichia coli uvrA recA bacteria exposed to a low dose (0.07 J/m2) of ultraviolet radiation was studied. A considerable amount of the newly-synthesized DNA, which contains gaps opposite pyrimidine dimers, is broken down. In contrast, parental, dimer-containing DNA is resistant to radiation-induced degradation.


Subject(s)
DNA Repair , DNA, Bacterial/radiation effects , Escherichia coli/radiation effects , Ultraviolet Rays , DNA Replication , DNA, Bacterial/metabolism , Radiation Dosage
12.
Nucleic Acids Res ; 12(3): 1563-72, 1984 Feb 10.
Article in English | MEDLINE | ID: mdl-6230599

ABSTRACT

The lysogenization of ultraviolet-irradiated Escherichia coli cells by the lambda phage was studied. Genetic analysis indicates that changes in the number of the lysogenized cell during post-UV growth is primarily due to the change in the proteolytic activity of RecA protein. Full proteolytic activity is achieved only in the presence of the functional recB gene product.


Subject(s)
Bacteriophage lambda/radiation effects , Escherichia coli Proteins , Escherichia coli/radiation effects , Exodeoxyribonucleases/genetics , Genes, Bacterial/radiation effects , Genes/radiation effects , Rec A Recombinases/radiation effects , Ultraviolet Rays , Bacteriophage lambda/genetics , Escherichia coli/genetics , Exodeoxyribonuclease V , Kinetics , Lysogeny
13.
Mol Gen Genet ; 196(1): 167-9, 1984.
Article in English | MEDLINE | ID: mdl-6237245

ABSTRACT

If ultraviolet irradiated, lambda-lysogenic Escherichia coli K12 bacteria are incubated for 4 to 6 h at 30 degrees C, lambda prophage becomes inactive in the non-surviving cells. This was demonstrated by the use of lambda cIts857ind1 prophage which can be induced by heat but not by ultraviolet light. An analysis with various bacterial mutants showed that RecBC recombination enzyme is required in conjunction with RecA protein for prophage inactivation.


Subject(s)
Bacteriophage lambda/radiation effects , Escherichia coli Proteins , Escherichia coli/genetics , Exodeoxyribonucleases/genetics , Lysogeny/radiation effects , DNA Repair , Exodeoxyribonuclease V , Hot Temperature , Rec A Recombinases/genetics , Ultraviolet Rays
15.
Mutat Res ; 112(1): 17-22, 1983 Feb.
Article in English | MEDLINE | ID: mdl-6338353

ABSTRACT

Post-UV DNA synthesis in Escherichia coli uvrA recA cells was studied. A low dose of UV radiation (0.07 J/m2), which caused no degradation of the dimer-containing DNA, was used. This enabled us to make a direct comparison between DNA synthesis on the normal template and DNA synthesis on the UV-damaged template. There was no change in the post-UV DNA synthesis kinetics during the first 60 min of post-irradiation incubation. A reduced rate of DNA synthesis was observed at later post-UV times when the dimers are expected to have passed through the normal replication complex. This reduced rate of DNA synthesis was associated with loss of the biological activity of the DNA. We suggest that the gaps opposite dimers rather than dimers per se interfere with normal replication, thus leading to cell death of uvrA recA bacteria.


Subject(s)
DNA Repair , DNA Replication , Escherichia coli/genetics , Pyrimidine Dimers/genetics , Bacterial Proteins/genetics , DNA, Bacterial/biosynthesis , Genes, Bacterial , Kinetics , Mutation , Rec A Recombinases , Ultraviolet Rays
16.
J Bacteriol ; 143(3): 1506-8, 1980 Sep.
Article in English | MEDLINE | ID: mdl-6997276

ABSTRACT

Deoxyribonucleic acid synthesis after ultraviolet irradiation was studied in wild-type, uvrA, recB, recA recB, and recA Escherichia coli strains. Inhibition of deoxyribonucleic acid synthesis, which occurs almost immediately after exposing the cells to ultraviolet radiation, depends on the functional gene recA.


Subject(s)
Bacterial Proteins/physiology , DNA, Bacterial/biosynthesis , Escherichia coli/metabolism , Recombination, Genetic , Escherichia coli/genetics , Escherichia coli/radiation effects , Ultraviolet Rays
19.
J Bacteriol ; 140(3): 848-51, 1979 Dec.
Article in English | MEDLINE | ID: mdl-160414

ABSTRACT

The fate of the prophage part of the lysogenic chromosome was followed in the course of post-ultraviolet incubation. For this purpose, lambda cI857 ind prophage, which can be induced by heat but not by ultraviolet light, was used. The prophage, intially more resistant than its repair-proficient host cell, was rapidly inactivated. This inactivation was not caused by the impaired capacity of irradiated cells to support growth of the phage. Over the entire dose range tested, little, if any, sensitivity difference between the host and the prophage was found at the end of cell division delay. Rapid inactivation of the prophage was also observed in uvr cells after small doses of ultraviolet light. The same small doses did not cause inactivation in lysogens carrying a mutation in the gene recA. This suggests that the functional gene recA is required for inactivation of the prophage part of the lysogenic chromosome.


Subject(s)
Bacteriophage lambda/growth & development , DNA Repair , Escherichia coli/genetics , Genes , Mutation , Recombination, Genetic , Bacteriophage lambda/radiation effects , Cell Survival/radiation effects , Escherichia coli/radiation effects , Hot Temperature , Lysogeny/radiation effects , Ultraviolet Rays , Virus Replication/radiation effects
20.
Mol Gen Genet ; 177(1): 91-4, 1979.
Article in English | MEDLINE | ID: mdl-161343

ABSTRACT

UV-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 c1857 ind red-lysogens were used. These lysogens, although non-inducible by UV light, can be induced by raising the temperature from 30 degrees to 42 degrees. If the W reactivation processes are involved in repair of the bacterial DNA, when the lysogens are incubated at 30 degrees 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.


Subject(s)
Chromosomes, Bacterial/radiation effects , DNA Repair , DNA, Bacterial/radiation effects , Escherichia coli/genetics , Ultraviolet Rays , Bacteriophage lambda , DNA, Viral , Escherichia coli/radiation effects , Lysogeny
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