Repair and recombination of nonreplicating UV-irradiated phage DNA in E. coli II. Stimulation of RecF-dependent recombination by excision repair of cyclobutane pyrimidine dimers and of other photoproducts

Summary Three aspects of recombination of UV-irradiated nonreplicating lambda phage DNA were addressed: the photoproduct(s) responsible, the role of UvrABC-mediated excision repair, and the dependence on RecF function.Cyclobutane pyrimidine dimers appeared responsible for some recombination because photoreactivation reduced the frequency of 254-nm-stimulated recombination and because photosensitized 313-nm irradiation stimulated recombination. Other photoproducts seemed recombinogenic as well, because high fluences of 254-nm irradiation stimulated recombination considerably more, per cyclobutane dimer induced, than photosensitized 313-nm irradiation, and because photoreactivation did not eliminate 254-nm stimulated recombination. For both treatments, much, but not all, of the recombination was UvrABC-dependent. Recombination was mostly RecF-dependent, but was not affected by recB recC or recE mutationsThe first paper in this series is Hays et al., (1985)     

Repair of nonreplicating UV-irradiated DNA: cooperative dark repair by Escherichia coli uvr and phr functions.

The system previously used to study recombination of nonreplicating UV-irradiated phage lambda DNA was adapted to study UV repair. Irradiated phages infected undamaged homoimmune lysogens. Pyrimidine dimer content (by treatment with Micrococcus luteus UV endonuclease and alkaline sucrose sedimentation) and a biological activity endpoint (infectivity in transfection of uvrB recA recB spheroplasts) were followed. Unless room light was excluded during DNA extraction procedures, photoreactivation (Phr function) was significant. In uvr delta phr bacteria, repair, by both assays, was very low but not zero. Even when light was totally excluded, Phr function appeared to play a role in Uvr-mediated excision repair: both dimer removal and restoration of infectivity were two to five times as efficient in uvr+ phr+ bacteria as in uvr+ delta phr bacteria. Similarly, UV-irradiated phages plated with higher efficiencies on phr+ than delta phr bacteria even under totally dark conditions. In uvr phr+ repressed infections, removal of dimers from nonreplicating DNA did not increase infectivity as much as in uvr+ infections, suggesting a requirement for repair of nondimer photoproducts by the uvrABC system.     

Rapid and apparently error-prone excision repair of nonreplicating UV-irradiated plasmids in Xenopus laevis oocytes.

Repair of UV-irradiated plasmid DNA microinjected into frog oocytes was measured by two techniques: transformation of repair-deficient (delta uvrB delta recA delta phr) bacteria, and removal of UV endonuclease-sensitive sites (ESS). Transformation efficiencies relative to unirradiated plasmids were used to estimate the number of lethal lesions; the latter were assumed to be Poisson distributed. These estimates were in good agreement with measurements of ESS. By both criteria, plasmid DNA was efficiently repaired, mostly during the first 2 h, when as many as 2 x 10(10) lethal lesions were removed per oocyte. This rate is about 10(6) times the average for removal of ESS from repair-proficient human cells. Repair was slower but still significant after 2 h, but some lethal lesions usually remained after overnight incubation. Most repair occurred in the absence of light, in marked contrast to differentiated frog cells, previously shown to possess photoreactivating but no excision repair activity. There was no increase in the resistance to DpnI restriction of plasmids (methylated in Escherichia coli at GATC sites) incubated in oocytes; this implies no increase in hemimethylated GATC sites, and hence no semiconservative DNA replication. Plasmid substrates capable of either intramolecular or intermolecular homologous recombination were not recombined, whether UV-irradiated or not. Repair of Lac+ plasmids was accompanied by a significant UV-dependent increase in the frequency of Lac- mutants, corresponding to a repair synthesis error frequency on the order of 10(-4) per nucleotide.     

Mismatch repair and recombination in E. coli

The involvement of the E. coli methyl-directed and very short patch (vsp) mismatch repair systems in bacteriophage lambda recombination has been studied. Genetic crosses and heteroduplex transfection experiments were performed using lambda phages with sequenced mutations in the cl gene. The results indicate that methyl-directed repair does operate during bacteriophage lambda recombination but generally does not contribute to the formation of recombinants involving close markers. Vsp repair apparently acts during bacteriophage lambda recombination to produce recombinants involving close markers because its action does not involve extensive excision tracts. Marker-specific hyperrecombination and the apparent clustering of genetic exchanges in bacteriophage lambda recombination can be accounted for by the action of the vsp repair system.     

Role of the bacterial and phage recombination systems and of DNA replication in genetic recombination of UV-irradiated phage lambda

Summary In this paper are studied in E. coli K12 the influence of the bacterial Rec and phage Red recombination systems on the rescue of the O ⁺ gene from the prophage by a superinfecting O ^- phage, UV irradiated or not. In the absence of UV irradiation the Red system produces more recombinants that does the Rec system, and its action requires DNA replication. The presence of UV lesions in the DNA facilitates the action of the Rec system, which is more efficient in this instance than the Red system and can act in the absence of DNA replication. In all cases, there is a cooperation between the two generalized recombination systems.     

RNA polymerase drives ribonucleotide excision DNA repair in E. coli

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Effects of puromycin aminonucleoside on excision repair and recombination repair inescherichia coli

Ultraviolet (UV) lethality was increased when puromycin aminonucleoside (PAN) (3.0 mM) was added to the postirradiation medium ofEscherichia coli strains. The extent of repair inhibition differed greatly for strains WP-2hcr ⁺, B/r()hcr ⁺, WP-2hcr ^–, and Bs-1hcr ^–. The interaction between PAN and UV was synergistic in thehcr ⁺ strains. PAN enhanced UV lethality in strain B/r () to a greater degree than in WP-2hcr ⁺. There was no UV lethality enhancement by PAN (3.0 mM) in thehcr ^– strains, but the interaction of PAN (8.0 mM) with UV was synergistic. PAN decreased plaque formation of T1 UV-irradiated phage plated onE. coli Bhcr ⁺ but had no effect on phage plated on Bs-1 or WP-2hcr ^– strains. These results suggest that PAN interferes with thehcr function in UV-irradiated bacteria.     

A model for the recA-dependent repair of excision gaps in UV-irradiated Escherichia coli

DNA isolated from lambda phage was treated with bleomycin A2 plus Fe2+. The bleomycin-damaged DNA was added to lambda packaging extracts and the resulting phage were grown in SOS-induced E. coli. Under these conditions, treatment of the DNA with 0.8 microM bleomycin reduced the viability of the repackaged phage to 3% and increased the frequency of clear-plaque mutants in the progeny by a factor of 16. Bleomycin-induced mutations which mapped to the DNA-binding domain of the cI gene were subjected to DNA-sequence analysis. The most frequent events were single-base substitutions at G:C base pairs, nearly all of which occurred at cytosines in the sequence Py-G-C. Cytosines in the third position of the sequence C-G-C-C were particularly susceptible to mutation. At A:T base pairs, mutations were less frequent and were a mixture of single-base substitutions and -1 frameshifts, occurring primarily at G-T and A-T sequences. Thus, the overall specificity of bleomycin-induced mutations matches that of bleomycin-induced DNA lesions (strand breaks and apyrimidinic sites), which are formed at G-C (particularly Py-G-C), G-T and, to a lesser extent, A-T sequences. Furthermore, the frequency of various types of substitutions was consistent with selective incorporation of A and T residues opposite apyrimidinic sites at these sequences. The highly selective nature of bleomycin-induced mutations may explain the lack of mutagenesis by this compound in a number of reversion assays.     

The modulating influence of the fluidity of cell membrane on excision repair of DNA in UV-irradiated Escherichia coli

When the extent of liquid holding recovery (LHR) was measured as a function of the temperature at the time of liquid holding and the Arrhenius plot was made, two distinctive phases for the LHR were demonstrated in UV-irradiated RecA- derivative of E. coli ole28E1, which are unable to synthesize and degrade unsaturated fatty acids. The inflection temperatures were 17-18 degrees C, 23-24 degrees C and 28-30 degrees C for linoleate-, oleate- and elaidate-grown cells, respectively. These temperatures well corresponded to the phase transition temperatures of the cell membrane supplemented with the fatty acid. It is therefore concluded that at least a component involved in in vivo excision repair in E. coli is associated with cell membrane.     

Repair of UV-irradiated plasmid DNA in mutants of Saccharomyces cerevisiae and Escherichia coli deficient in repair of pyrimidine dimers

The repair of in vitro UV-irradiated DNA of plasmid pBB29 was studied in excision defective yeast mutants rad1, rad2, rad3, rad4, rad10 and in Escherichia coli mutants uvr- and recA-, by measuring the cell transformation frequency. Rad2, rad3, rad4, and rad10 mutants could repair plasmid DNA despite their inability to repair nuclear DNA, whereas the reduced ability of rad1 mutant for plasmid DNA repair demonstrated alone the same dependence on the host functions that are needed for nuclear DNA repair. In E. coli the repair of UV-irradiated plasmid DNA is carried out only by the excision-repair system dependent on uvr genes. Treatment of UV-irradiated plasmid DNA with UV endonuclease from Micrococcus luteus greatly enhances the efficiency of transformation of E. coli uvr- mutants. Similar treatment with cell-free extracts of yeast rad1 mutant or wild-type strains as well as with nuclease BaL31, despite their ability for preferential cutting of UV damaged DNA, showed no influence on cell transformation.     

Role of the E. coli umuC gene product in the repair of single-stranded DNA phage

The umuC product of Escherichia coli has been suggested to have a central role in SOS induced error prone replication of DNA (Kato and Shinoura 1977). To investigate this possibility, we examined the effect of umuC mutations on error prone repair of single and double-stranded DNA phages. No Weigle reactivation of M13 phage was detected in a umuC mutant. Reactivation of lambda phage was reduced but still evident. However mutagenesis occurred in both cases. These results suggest that induced error prone replication of phage DNA can occur via umuC dependent (transdimer synthesis) and umuC independent mechanisms.     

Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli.

Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes. (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected. These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein. However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair. Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA.     

Functions of base flipping in E. coli nucleotide excision repair

UvrB is the main damage recognition protein in bacterial nucleotide excision repair and is capable of recognizing various structurally unrelated types of damage. Previously we have shown that upon binding of Escherichia coli UvrB to damaged DNA two nucleotides become extrahelical: the nucleotide directly 3' to the lesion and its base-pairing partner in the non-damaged strand. Here we demonstrate using a novel fluorescent 2-aminopurine-menthol modification that the position of the damaged nucleotide itself does not change upon UvrB binding. A co-crystal structure of B. caldotenax UvrB and DNA has revealed that one nucleotide is flipped out of the DNA helix into a pocket of the UvrB protein where it stacks on Phe249 [J.J. Truglio, E. Karakas, B. Hau, H. Wang, M.J. DellaVecchia, B. van Houten, C. Kisker, Structural basis for DNA recognition and processing by UvrB, Nat. Struct. Mol. Biol. 13 (2006) 360-364]. By mutating the equivalent of Phe249 (Tyr249) in the E. coli UvrB protein we show that on damaged DNA neither of the extrahelical nucleotides is inserted into this protein pocket. The mutant UvrB protein, however, resulted in an increased binding and incision of undamaged DNA showing that insertion of a base into the nucleotide-binding pocket is important for dissociation of UvrB from undamaged sites. Replacing the nucleotides in the non-damaged strand with a C3-linker revealed that the extruded base in the non-damaged strand is not directly involved in UvrB-binding or UvrC-mediated incision, but that its displacement is needed to allow access for residues of UvrB or UvrC to the neighboring base, which is directly opposite the DNA damage. This interaction is shown to be essential for optimal 3'-incision by UvrC. After 3'-incision base flipping in the non-damaged DNA strand is lost, indicative for a conformational change needed to prepare the UvrB-DNA complex for 5'-incision.     

Nucleotide excision repair: from E. coli to man

Nucleotide excision repair is both a 'wide spectrum' DNA repair pathway and the sole system for repairing bulky damages such as UV lesions or benzo[a]pyrene adducts. The mechanisms of nucleotide excision repair are known in considerable detail in Escherichia coli. Similarly, in the past 5 years important advances have been made towards understanding the biochemical mechanisms of excision repair in humans. The overall strategy of the repair is the same in the two species: damage recognition through a multistep mechanism involving a molecular matchmaker and an ATP-dependent unwinding of the damaged duplex; dual incisions at both sides of the lesion by two different nucleases, the 3' incision being followed by the 5'; removal of the damaged oligomer; resynthesis of the repair patch, whose length matches the gap size. Despite these similarities, the two systems are biochemically different and do not even share structural homology. E. coli excinuclease employs three proteins in contrast to 16/17 polypeptides in man; the excised fragment is longer in man: the procaryotic excinuclease is not able by itself to remove the excised oligomer whereas the human enzyme does. Thus, the excinuclease mode of action is well conserved throughout evolution, but not the biochemical tools: this represents a case of evolutionary convergence.     

Effect of some drugs on excision repair in E. coli cells.

The intercalating dye ethidium bromide (EB), inhibits excision of pyrimidine dimers from UV-irradiated excision-proficient Escherichia coli B/r hcr+ cells. Inhibition is total at a 2.5 - 10(-4) M concentration 120 min after irradiation with a dose of 750 erg/mm2. The viability of irradiated cells diminishes in proportion to the EB concentration. Under wholly analogous conditions of cultivation and irradiation no inhibitory effect of KCN and caffeine (CFF) and only a slight effect of chloramphenicol (CAP) on dimer excision has been observed. The viability of cells is affected by these compounds but it does not appear to depend on the quantity of excised photoproducts. A change in the secondary structure of DNA induced by intercalation of EB appears to be the reason for the depression of excision of UV photoproducts.     

2-aminopurine induced DNA repair in E. coli

Summary The survival of UV-irradiated phages is increased when host bacteria are grown in the presence of the base analogue 2-aminopurine (2AP) before infection. This increase in survival, which we have called 2AP-reactivation depends upon the concentration of 2AP and the time of exposure to 2AP. 2AP-reactivation can be distinguished from Weigle-reactivation in that it is not accompanied by an increase in mutagenesis, does not act on the single-stranded DNA bacteriophage X174, and occurs in recA and lexA bacteria. 2AP reactivation does not appear to involve known systems of recombinational repair, as it occurs in recB and recF bacteria, or excision repair, as it occurs in uvrA and uvrB bacteria. It is however dependent upon DNA polymerase I.