Collapse and repair of replication forks in Escherichia coli

Single-strand interruptions in a template DNA are likely to cause collapse of replication forks. We propose a model for the repair of collapsed replication forks in Escherichia coli by the RecBCD recombinational pathway. The model gives reasons for the preferential orientation of Chi sites in the E. coli chromosome and accounts for the hyper-rec phenotype of the strains with increased numbers of single-strand interruptions in their DNA. On the basis of the model we offer schemes for various repeat-mediated recombinational events and discuss a mechanism for quasi-conservative DNA replication explaining the recombinational repair-associated mutagenesis.     

Rat DNA polymerase beta gene can join in excision repair of Escherichia coli.

Though DNA polymerase I (poll) of Escherichia (E.) coli is understood to play a role in repair synthesis of excision repair, it is still obscure whether DNA polymerase beta (pol beta) plays a similar role in eukaryotic cells. To estimate the role of pol beta in excision repair processes, we inserted the rat pol beta gene into several mutant E. coli defective in a diverse set of enzymatic activities of poll. UV resistance was seen only when the 5'----3' exonuclease (exo) activity of poll molecules remained. Therefore it is suggested that 5'----3' exo activity as well as pol beta activity are essential for repair synthesis of excision repair in eukaryotic cells.     

Role of the Escherichia coli nucleotide excision repair proteins in DNA replication

DNA polymerase I (PolI) functions both in nucleotide excision repair (NER) and in the processing of Okazaki fragments that are generated on the lagging strand during DNA replication. Escherichia coli cells completely lacking the PolI enzyme are viable as long as they are grown on minimal medium. Here we show that viability is fully dependent on the presence of functional UvrA, UvrB, and UvrD (helicase II) proteins but does not require UvrC. In contrast, delta polA cells grow even better when the uvrC gene has been deleted. Apparently UvrA, UvrB, and UvrD are needed in a replication backup system that replaces the PolI function, and UvrC interferes with this alternative replication pathway. With specific mutants of UvrC we could show that the inhibitory effect of this protein is related to its catalytic activity that on damaged DNA is responsible for the 3' incision reaction. Specific mutants of UvrA and UvrB were also studied for their capacity to support the PolI-independent replication. Deletion of the UvrC-binding domain of UvrB resulted in a phenotype similar to that caused by deletion of the uvrC gene, showing that the inhibitory incision activity of UvrC is mediated via binding to UvrB. A mutation in the N-terminal zinc finger domain of UvrA does not affect NER in vivo or in vitro. The same mutation, however, does give inviability in combination with the delta polA mutation. Apparently the N-terminal zinc-binding domain of UvrA has specifically evolved for a function outside DNA repair. A model for the function of the UvrA, UvrB, and UvrD proteins in the alternative replication pathway is discussed.     

dnaC-dependent reconstitution of replication forks in Escherichia coli lysates.

Lysates of Escherichia coli exhibit a DNA-synthesizing activity that depends on the presence of replication forks and of replication proteins. Replicative activity was reconstituted in vitro by mixing lysates prepared from temperature-sensitive dnaB mutants with wild-type dnaB protein. Lysates of double mutants deficient in both dnaB and dnaC genes could only be complemented by the addition of both dnaB and dnaC proteins, whereas lysates deficient in dnaC protein did not require the addition of any exogenous factor. This shows that the replication machinery, once it is running along the chromosome, is independent of dnaC protein, dnaC activity, however, is required for the replacement of defective dnaB protein at running replication forks.     

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.     

Mammalian DNA polymerase beta can substitute for DNA polymerase I during DNA replication in Escherichia coli.

Mammalian DNA polymerase beta is the smallest known eukaryotic polymerase and is expressed as an active protein in Escherichia coli harboring a plasmid containing its cDNA. Since some catalytic functions of DNA polymerase beta and E. coli DNA polymerase I are similar, we wished to determine if DNA polymerase beta could substitute for DNA polymerase I in bacteria. We found that the expression of mammalian DNA polymerase beta in E. coli restored growth in a DNA polymerase I-defective bacterial mutant. Sucrose density gradient analysis revealed that DNA polymerase beta complements the replication defect in the mutant by increasing the rate of joining of Okazaki fragments. These findings demonstrate that DNA polymerase beta, believed to function in DNA repair in mammalian cells, can also function in DNA replication. Moreover, this complementation system will permit study of the in vivo function of altered species of DNA polymerase beta, an analysis currently precluded by the difficulty in isolating mutants in mammalian cells.     

RecQ and RecJ process blocked replication forks prior to the resumption of replication in UV-irradiated Escherichia coli

The accurate recovery of replication following DNA damage and repair is critical for the maintenance of genomic integrity. In Escherichia coli, the recovery of replication following UV-induced DNA damage is dependent upon several proteins in the recF pathway, including RecF, RecO, and RecR. Two other recF pathway proteins, the RecQ helicase and the RecJ exonuclease, have been shown to affect the sites and frequencies at which illegitimate rearrangements occur following UV-induced DNA damage, suggesting that they also may function during the recovery of replication. We show here that RecQ and RecJ process the nascent DNA at blocked replication forks prior to the resumption of DNA synthesis. The processing involves selective degradation of the nascent lagging DNA strand and it requires both RecQ and RecJ. We suggest that this processing may serve to lengthen the substrate that can be recognized and stabilized by the RecA protein at the replication fork, thereby helping to ensure the accurate recovery of replication after the obstructing lesion has been repaired. Received: 1 June 1999 / Accepted: 28 July 1999     

Nucleotide excision repair and recombination are engaged in repair of trans-4-hydroxy-2-nonenal adducts to DNA bases in Escherichia coli

One of the major products of lipid peroxidation is trans-4-hydroxy-2-nonenal (HNE). HNE forms highly mutagenic and genotoxic adducts to all DNA bases. Using M13 phage lacZ system, we studied the mutagenesis and repair of HNE treated phage DNA in E. coli wild-type or uvrA, recA, and mutL mutants. These studies revealed that: (i) nucleotide excision and recombination, but not mismatch repair, are engaged in repair of HNE adducts when present in phage DNA replicating in E. coli strains; (ii) in the single uvrA mutant, phage survival was drastically decreased while mutation frequency increased, and recombination events constituted 48 % of all mutations; (iii) in the single recA mutant, the survival and mutation frequency of HNE-modified M13 phage was slightly elevated in comparison to that in the wild-type bacteria. The majority of mutations in recA^- strain were G:C → T:A transversions, occurring within the sequence which in recA⁺ strains underwent RecA-mediated recombination, and the entire sequence was deleted; (iv) in the double uvrA recA mutant, phage survival was the same as in the wild-type although the mutation frequency was higher than in the wild-type and recA single mutant, but lower than in the single uvrA mutant. The majority of mutations found in the latter strain were base substitutions, with G:C → A:T transitions prevailing. These transitions could have resulted from high reactivity of HNE with G and C, and induction of SOS-independent mutations.     

The Escherichia coli preprimosome and DNA B helicase can form replication forks that move at the same rate

A DNA replication system was developed that could generate rolling-circle DNA molecules in vitro in amounts that permitted kinetic analyses of the movement of the replication forks. Two artificial primer-template DNA substrates were used to study DNA synthesis catalyzed by the DNA polymerase III holoenzyme in the presence of either the preprimosomal proteins (the primosomal proteins minus the DNA G primase) and the Escherichia coli single-stranded DNA binding protein or the DNA B helicase alone. Helicase activities have recently been demonstrated to be associated with the primosome, a mobile multiprotein priming apparatus that requires seven E. coli proteins (replication factor Y (protein n'), proteins n and n', and the products of the dnaB, dnaC, dnaG, and dnaT genes) for assembly, and with the DNA B protein. Consistent with a rolling-circle mechanism in which a helicase activity permitted extensive (-) strand DNA synthesis on a (+) single-stranded, circular DNA template, the major DNA products formed were multigenome-length, single-stranded, linear molecules. The replication forks assembled with either the preprimosome or the DNA B helicase moved at the same rate (approximately 730 nucleotides/s) at 30 degrees C and possessed apparent processivities in the range of 50,000-150,000 nucleotides. The single-stranded DNA binding protein was not required to maintain this high rate of movement in the case of leading strand DNA synthesis catalyzed by the DNA polymerase III holoenzyme and the DNA B helicase.     

Persistently stalled replication forks inhibit nucleotide excision repair in trans by sequestering Replication protein A

Rev3, the catalytic subunit of DNA polymerase ζ, is essential for translesion synthesis of cytotoxic DNA photolesions, whereas the Rev1 protein plays a noncatalytic role in translesion synthesis. Here, we reveal that mammalian Rev3^−/− and Rev1^−/− cell lines additionally display a nucleotide excision repair (NER) defect, specifically during S phase. This defect is correlated with the normal recruitment but protracted persistence at DNA damage sites of factors involved in an early stage of NER, while repair synthesis is affected. Remarkably, the NER defect becomes apparent only at 2 h post-irradiation indicating that Rev3 affects repair synthesis only indirectly, rather than performing an enzymatic role in NER. We provide evidence that the NER defect is caused by scarceness of Replication protein A (Rpa) available to NER, resulting from its sequestration at stalled replication forks. Also the induction of replicative stress using hydroxyurea precludes the accumulation of Rpa at photolesion sites, both in Rev3^−/− and in wild-type cells. These data support a model in which the limited Rpa pool coordinates replicative stress and NER, resulting in increased cytotoxicity of ultraviolet light when replicative stress exceeds a threshold.     

Multiple genetic pathways for restarting DNA replication forks in Escherichia coli K-12

In Escherichia coli, the primosome assembly proteins, PriA, PriB, PriC, DnaT, DnaC, DnaB, and DnaG, are thought to help to restart DNA replication forks at recombinational intermediates. Redundant functions between priB and priC and synthetic lethality between priA2::kan and rep3 mutations raise the possibility that there may be multiple pathways for restarting replication forks in vivo. Herein, it is shown that priA2::kan causes synthetic lethality when placed in combination with either Deltarep::kan or priC303:kan. These determinations were made using a nonselective P1 transduction-based viability assay. Two different priA2::kan suppressors (both dnaC alleles) were tested for their ability to rescue the priA-priC and priA-rep double mutant lethality. Only dnaC809,820 (and not dnaC809) could rescue the lethality in each case. Additionally, it was shown that the absence of the 3'-5' helicase activity of both PriA and Rep is not the critical missing function that causes the synthetic lethality in the rep-priA double mutant. One model proposes that replication restart at recombinational intermediates occurs by both PriA-dependent and PriA-independent pathways. The PriA-dependent pathways require at least priA and priB or priC, and the PriA-independent pathway requires at least priC and rep. It is further hypothesized that the dnaC809 suppression of priA2::kan requires priC and rep, whereas dnaC809,820 suppression of priA2::kan does not.     

Role of high-fidelity Escherichia coli DNA polymerase I in replication bypass of a deoxyadenosine DNA-peptide cross-link

Reaction of bifunctional electrophiles with DNA in the presence of peptides can result in DNA-peptide cross-links. In particular, the linkage can be formed in the major groove of DNA via the exocyclic amino group of adenine (N⁶-dA). We previously demonstrated that an A family human polymerase, Pol ν, can efficiently and accurately synthesize DNA past N⁶-dA-linked peptides. Based on these results, we hypothesized that another member of that family, Escherichia coli polymerase I (Pol I), may also be able to bypass these large major groove DNA lesions. To test this, oligodeoxynucleotides containing a site-specific N⁶-dA dodecylpeptide cross-link were created and utilized for in vitro DNA replication assays using E. coli DNA polymerases. The results showed that Pol I and Pol II could efficiently and accurately bypass this adduct, while Pol III replicase, Pol IV, and Pol V were strongly inhibited. In addition, cellular studies were conducted using E. coli strains that were either wild type or deficient in all three DNA damage-inducible polymerases, i.e., Pol II, Pol IV, and Pol V. When single-stranded DNA vectors containing a site-specific N⁶-dA dodecylpeptide cross-link were replicated in these strains, the efficiencies of replication were comparable, and in both strains, intracellular bypass of the lesion occurred in an error-free manner. Collectively, these findings demonstrate that despite its constrained active site, Pol I can catalyze DNA synthesis past N⁶-dA-linked peptide cross-links and is likely to play an essential role in cellular bypass of large major groove DNA lesions.     

Strand specificity of ribonucleotide excision repair in Escherichia coli

In Escherichia coli, replication of both strands of genomic DNA is carried out by a single replicase—DNA polymerase III holoenzyme (pol III HE). However, in certain genetic backgrounds, the low-fidelity TLS polymerase, DNA polymerase V (pol V) gains access to undamaged genomic DNA where it promotes elevated levels of spontaneous mutagenesis preferentially on the lagging strand. We employed active site mutants of pol III (pol IIIα_S759N) and pol V (pol V_Y11A) to analyze ribonucleotide incorporation and removal from the E. coli chromosome on a genome-wide scale under conditions of normal replication, as well as SOS induction. Using a variety of methods tuned to the specific properties of these polymerases (analysis of lacI mutational spectra, lacZ reversion assay, HydEn-seq, alkaline gel electrophoresis), we present evidence that repair of ribonucleotides from both DNA strands in E. coli is unequal. While RNase HII plays a primary role in leading-strand Ribonucleotide Excision Repair (RER), the lagging strand is subject to other repair systems (RNase HI and under conditions of SOS activation also Nucleotide Excision Repair). Importantly, we suggest that RNase HI activity can also influence the repair of single ribonucleotides incorporated by the replicase pol III HE into the lagging strand.     

RecA-mediated rescue of Escherichia coli strains with replication forks arrested at the terminus

The recombinational rescue of chromosome replication was investigated in Escherichia coli strains with the unidirectional origin oriR1, from the plasmid R1, integrated within oriC in clockwise (intR1(CW)) or counterclockwise (intR1(CC)) orientations. Only the intR1(CC) strain, with replication forks arrested at the terminus, required RecA for survival. Unlike the strains with RecA-dependent replication known so far, the intR1(CC) strain did not require RecBCD, RecF, RecG, RecJ, RuvAB, or SOS activation for viability. The overall levels of degradation of replicating chromosomes caused by inactivation of RecA were similar in oriC and intR1(CC) strains. In the intR1(CC) strain, RecA was also needed to maintain the integrity of the chromosome when the unidirectional replication forks were blocked at the terminus. This was consistent with suppression of the RecA dependence of the intR1(CC) strain by inactivating Tus, the protein needed to block replication forks at Ter sites. Thus, RecA is essential during asymmetric chromosome replication for the stable maintenance of the forks arrested at the terminus and for their eventual passage across the termination barrier(s) independently of the SOS and some of the major recombination pathways.     

Dimer excision and repair replication patch size in recL152 mutant of Escherichia coli K-12.

Dimers are excised slowly in a recL152 mutant. This observation is not an artifact of altered DNA degradation because degradation is the same in recL+ and recL strains. The repair patch size was measured by the bromodeoxyuridine-313 nm radiation photolysis technique. In the recL+ strain, the average patch size was found to be about 30 nucleotides in length, but in the recL mutant, it was about 360.     

Two modes of excision repair in toluene-treated Escherichia coli.

In toluene-treated Escherichia coli incision breaks accumulate during post-irradiation incubation in the presence of adenosine 5'-triphosphate (ATP). It is shown that incised deoxyribonucleic acid (DNA) is converted to high-molecular-weight DNA during reincubation in the presence of the four deoxyribonucleoside triphosphates (dNTP's) and nicotinamide adenine dinucleotide (NAD). This restitution process is ATP independent and N-ethylmaleimide insensitive and takes place only in polA+ strains. It is defective in strains carrying a mutation in the 5' leads to 3' exonucleolytic activity associated with DNA polymerase I. Repair of accumulated incision breaks differs from repair in which all the steps of the excision repair process occur simultaneously or in rapid succession. The latter is observed if toluene-treated E. coli are incubated immediately after irradiation in the presence of the four dNTP's, NAD, and ATP. It is shown that under these conditions dimer excision occurs to a larger extent than during repair of accumulated incision breaks and that, except in strains defective in polynucleotide ligase, incision breaks do not accumulate. This consecutive mode of repair is detectable in polA+ strains and at low doses also in polA mutants.     

The excision of 3′ penultimate errors by DNA polymerase I and its role in endonuclease V-mediated DNA repair

Deamination of adenine can occur spontaneously under physiological conditions, and is enhanced by exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat, generating the highly mutagenic lesion of deoxyinosine in DNA. Such DNA lesions tends to generate A:T to G:C transition mutations if unrepaired. In Escherichia coli, deoxyinosine is primarily removed through a repair pathway initiated by endonuclease V (endo V). In this study, we compared the repair of three mutagenic deoxyinosine lesions of A-I, G-I, and T-I using E. coli cell-free extracts as well as reconstituted protein system. We found that 3′-5′ exonuclease activity of DNA polymerase I (pol I) was very important for processing all deoxyinosine lesions. To understand the nature of pol I in removing damaged nucleotides, we systemically analyzed its proofreading to 12 possible mismatches 3′-penultimate of a nick, a configuration that represents a repair intermediate generated by endo V. The results showed all mismatches as well as deoxyinosine at the 3′ penultimate site were corrected with similar efficiency. This study strongly supports for the idea that the 3′-5′ exonuclease activity of E. coli pol I is the primary exonuclease activity for removing 3′-penultimate deoxyinosines derived from endo V nicking reaction.