Oligomerization of the UvrB nucleotide excision repair protein of Escherichia coli.

A combination of hydrodynamic and cross-linking studies were used to investigate self-assembly of the Escherichia coli DNA repair protein UvrB. Though the procession of steps leading to incision of DNA at sites flanking damage requires that UvrB engage in an ordered series of complexes, successively with UvrA, DNA, and UvrC, the potential for self-association had not yet been reported. Gel permeation chromatography, nondenaturing polyacrylamide gel electrophoresis, and chemical cross-linking results combine to show that UvrB stably assembles as a dimer in solution at concentrations in the low micromolar range. Smaller populations of higher order oligomeric species are also observed. Unlike the dimerization of UvrA, an initial step promoted by ATP binding, the monomer-dimer equilibrium for UvrB is unaffected by the presence of ATP. The insensitivity of cross-linking efficiency to a 10-fold variation in salt concentration further suggests that UvrB self-assembly is driven largely by hydrophobic interactions. Self-assembly is significantly weakened by proteolytic removal of the carboxyl terminus of the protein (generating UvrB*), a domain also known to be required for the interaction with UvrC leading to the initial incision of damaged DNA. This suggests that the C terminus may be a multifunctional binding domain, with specificity regulated by protein conformation.     

Interaction between the mismatch repair and nucleotide excision repair pathways in the prevention of 5-azacytidine-induced CG-to-GC mutations in Escherichia coli

5-Azacytidine induces CG-to-GC transversion mutations in Escherichia coli. The results presented in this paper provide evidence that repair of the drug-induced lesions that produce these mutations involves components of both the mismatch repair and nucleotide excision repair systems. Strains deficient in mutL, mutS, uvrA, uvrB or uvrC all showed an increase in mutation in response to 5-azacytidine. Using a bacterial two-hybrid assay, we showed that UvrB interacts with MutL and MutS in a drug-dependent manner, while UvrC interacts with MutL independent of drug. We suggest that 5-azacytidine-induced mismatches recruit MutS and MutL, but are poorly processed by mismatch repair. Instead, the stalled MutS–MutL complex recruits the Uvr proteins to complete repair.     

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.     

Hierarchy of DNA damage recognition in Escherichia coli nucleotide excision repair

DNA damage recognition plays a central role in nucleotide excision repair (NER). Here we present evidence that in Escherichia coli NER, DNA damage is recognized through at least two separate but successive steps, with the first focused on distortions from the normal structure of the DNA double helix (initial recognition) and the second specifically recognizing the type of DNA base modifications (second recognition), after an initial local separation of the DNA strands. DNA substrates containing stereoisomeric (+)- or (-)-trans- or (+)- or (-)-cis-BPDE-N(2)-dG lesions in DNA duplexes of known conformations were incised by UvrABC nuclease with efficiencies varying by up to 3-fold. However, these stereoisomeric adducts, when positioned in an opened, single-stranded DNA region, were all incised with similar efficiencies and with enhanced rates (by factors of 1.4-6). These bubble substrates were also equally and efficiently incised by UvrBC nuclease without UvrA. Furthermore, removal of the Watson-Crick partner cytosine residue (leaving an abasic site) in the complementary strand opposite a (+)-cis-BPDE-N(2)-dG lesion led to a significant reduction in both the binding of UvrA and the incision efficiency of UvrABC by a factor of 5. These data suggest that E. coli NER features a dynamic two-stage recognition mechanism.     

(A)BC excinuclease: the Escherichia coli nucleotide excision repair enzyme

Nucleotide excision repair is the major pathway for removing damage from DNA. (A)BC excinuclease is the nuclease activity which initiates nucleotide excision repair in Escherichia coli. In this review, we focus on current understanding of the structure-function of the enzyme and the reaction mechanism of the repair pathway. In addition, recent biochemical studies on preferential repair of actively transcribed genes in E. coli are summarized.     

Overexpression of Escherichia coli nucleotide excision repair genes after cisplatin-induced damage

Cisplatin is currently used in tumor chemotherapy to induce the death of malignant cells through blockage of DNA replication. It is a commonly used chemotherapeutic agent binding mono- or bifunctionally to guanines in DNA. Escherichia coli K12 mutant strains deficient in nucleotide excision repair (NER) were submitted to increasing concentrations of cisplatin, and the results revealed that uvrA and uvrB mutants are sensitive to this agent, while uvrC and cho mutants remain as the wild type strain. The time required for both gene expression turn-off and return to normal weight DNA in wild-type E. coli was not accomplished even after 4 h post-treatment with cisplatin, while the same process takes place within 1.5 h after ultraviolet radiation (UV). Besides, a heavily damaging action of cisplatin can be seen not only by persistent nicks on genomic DNA, but also by NER gene expression exceeding manifold that seen after equivalent lethal doses of UV. Moreover, cisplatin caused an increase in uvrB gene expression from its putative upstream promoter P3 in an SOS-independent manner.     

Both ATPase sites of Escherichia coli UvrA have functional roles in nucleotide excision repair.

The roles of the two tandemly arranged putative ATP binding sites of Escherichia coli UvrA in UvrABC endonuclease-mediated excision repair were analyzed by site-directed mutagenesis and biochemical characterization of the representative mutant proteins. Evidence is presented that UvrA has two functional ATPase sites which coincide with the putative ATP binding motifs predicted from its amino acid sequence. The individual ATPase sites can independently hydrolyze ATP. The C-terminal ATPase site has a higher affinity for ATP than the N-terminal site. The invariable lysine residues at the ends of the glycine-rich loops of the consensus Walker type "A" motifs are indispensable for ATP hydrolysis. However, the mutations at these lysine residues do not significantly affect ATP binding. UvrA, with bound ATP, forms the most favored conformation for DNA binding. The initial binding of UvrA to DNA is chiefly at the undamaged sites. In contrast to the wild type UvrA, the ATPase site mutants bind equally to damaged and undamaged sites. Dissociation of tightly bound nucleoprotein complexes from the undamaged sites requires hydrolysis of ATP by the C-terminal ATPase site of UvrA. Thus, both ATP binding and hydrolysis are required for the damage recognition step enabling UvrA to discriminate between damaged and undamaged sites on DNA.     

Interactions between yeast photolyase and nucleotide excision repair proteins in Saccharomyces cerevisiae and Escherichia coli.

The PHR1 gene of Saccharomyces cerevisiae encodes a DNA photolyase that catalyzes the light-dependent repair of pyrimidine dimers. In the absence of photoreactivating light, this enzyme binds to pyrimidine dimers but is unable to repair them. We have assessed the effect of bound photolyase on the dark survival of yeast cells carrying mutations in genes that eliminate either nucleotide excision repair (RAD2) or mutagenic repair (RAD18). We found that a functional PHR1 gene enhanced dark survival in a rad18 background but failed to do so in a rad2 or rad2 rad18 background and therefore conclude that photolyase stimulates specifically nucleotide excision repair of dimers in S. cerevisiae. This effect is similar to the effect of Escherichia coli photolyase on excision repair in the bacterium. However, despite the functional and structural similarities between yeast photolyase and the E. coli enzyme and complementation of the photoreactivation deficiency of E. coli phr mutants by PHR1, yeast photolyase failed to enhance excision repair in the bacterium. Instead, Phr1 was found to be a potent inhibitor of dark repair in recA strains but had no effect in uvrA strains. The results of in vitro experiments indicate that inhibition of nucleotide excision repair results from competition between yeast photolyase and ABC excision nuclease for binding at pyrimidine dimers. In addition, the A and B subunits of the excision nuclease, when allowed to bind to dimers before photolyase, suppressed photoreactivation by Phr1. We propose that enhancement of nucleotide excision repair by photolyases is a general phenomenon and that photolyase should be considered an accessory protein in this pathway.     

Recombinant expression of maize nucleotide excision repair protein Rad23 in Escherichia coli

Nucleotide excision is a highly conserved DNA repair pathway for correcting DNA lesions that cause distortion of the double helical structure. The protein heterodimer XPC-Rad23 is involved in recognition of and binding to such lesions. We have isolated full-length cDNAs encoding two different members of the maize Rad23 family. The deduced amino acid sequences of both maize orthologues show a high degree of homology to plant and animal Rad23 proteins. The cDNA encoding maize Rad23A was cloned as an in-frame C-terminal fusion of glutathione S-transferase. This chimera was expressed in Escherichia coli as a soluble protein and purified to homogeneity using glutathione-agarose followed by MonoQ column chromatography. Purified recombinant maize Rad23 protein was used to generate polyclonal antibodies that cross-react with a approximately 48-kDa protein in extracts from plant as well as mammalian cells. The purified recombinant protein and antibodies would be useful reagents to study the biochemistry of nucleotide excision repair in plants.     

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.     

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.     

In vivo formation and repair of cyclobutane pyrimidine dimers and 6-4 photoproducts measured at the gene and nucleotide level in Escherichia coli

In vivo formation and repair of the major UV-induced DNA photoproducts, cyclobutane pyrimidine dimers (CPDs) and 6-4 pyrimidine-pyrimidone photoproducts (6-4 PPs), have been examined at the gene and nucleotide level in Escherichia coli. Each type of DNA photoproduct has individually been studied using photoreactivation and two newly developed assays; the multiplex QPCR assay for damage detection at the gene level and the reiterative primer extension (PE) assay for damage detection at the nucleotide level. In the E. coli lacI and lacZ genes, CPDs and 6-4 PPs form in a 2:1 ratio, respectively, during UV irradiation. Repair of 6-4 PPs is more efficient than repair of CPDs since, on the average, 42% of 6-4 PPs are repaired in both genes in the first 40 min following 200 J/m² UV irradiation, while 1% of CPDs are repaired. The location, relative frequency of formation, and efficiency of repair of each type of photoproduct was examined in the first 52 codons of the E. coli lacI gene at the nucleotide level. Hotspots of formation were found for each type of lesion. Most photoproducts are at sites where both CPDs and 6-4 PPs are formed. Allowing 40 min of recovery following 200 J/m² shows that in vivo repair of 6-4 PPs is about fourfold more efficient than the repair of CPDs. Comparison of the lesion-specific photoproduct distribution of the lacI gene with a UV-induced mutation spectrum from wild-type cells shows that most mutational hotspots are correlated with sites of a majority of CPD formation. However, 6-4 PPs are also formed at some of these sites with relatively high frequency. This information, taken together with the observation that 6-4 PPs are repaired faster than CPDs, suggest that the cause of mutagenic hotspots in wild-type E. coli is inefficient repair of CPDs.     

The role of nucleotide excision repair of Escherichia coli in repair of spontaneous and gamma-radiation-induced DNA damage in the lacZalpha gene

Base excision repair (BER) is a very important repair mechanism to remove oxidative DNA damage. A major oxidative DNA damage after exposure to ionizing radiation is 7,8-dihydro-8-oxoguanine (8oxoG). 8oxoG is a strong mutagenic lesion, which may cause G:C to T:A transversions if not repaired correctly. Formamidopyrimidine-DNA glycosylase (Fpg), a repair enzyme which is part of BER, is the most important enzyme to repair 8oxoG. In the past years, evidence evolved that nucleotide excision repair (NER), a repair system originally thought to repair only bulky DNA lesions, can also repair some oxidative DNA damages. Examples of DNA damages which are recognized by NER are thymine glycol and abasic sites (AP sites). The main objective of this study is to determine if NER can act as a backup system for the repair of spontaneous and gamma-radiation-induced damages when Fpg is deficient. For that purpose, the effect of a NER-deficiency on the spontaneous and gamma-radiation-induced mutation spectrum in the lacZ gene was determined, using double-stranded (ds) M13 DNA, with the lacZalpha gene inserted as mutational target sequence. Subsequently the DNA was transfected into a fpg(-)uvrA(-) Escherichia coli strain (BH420) and the mutational spectra were compared with the spectra of a fpg(-) E. coli strain (BH410) and a wild type E. coli strain (JM105), which were determined in an earlier study. Furthermore, to examine effects which are caused by UvrA-deficiency, and not by Fpg-deficiency, the spontaneous and gamma-radiation-induced mutation spectra of an E. coli strain in which only UvrA is deficient (BH430) were also determined and compared with a wild type E. coli strain (JM105). The results of this study indicate that if only UvrA is deficient, there is an increase in spontaneous G:C to T:A transversions as compared to JM105 and a decrease in A:T to G:C transitions. The gamma-radiation-induced mutation spectrum of BH420 (fpg(-)uvrA(-)) shows a significant decrease in G:C to A:T and G:C to T:A mutations, as compared to BH410 where only Fpg is deficient. Based on these results, we conclude that in our experiments NER is not acting as a backup system if Fpg is deficient. Instead, NER seems to make mistakes, leading to the formation of mutations.     

Involvement of the nucleotide excision repair protein UvrA in instability of CAG*CTG repeat sequences in Escherichia coli.

Several human genetic diseases have been associated with the genetic instability, specifically expansion, of trinucleotide repeat sequences such as (CTG)(n).(CAG)(n). Molecular models of repeat instability imply replication slippage and the formation of loops and imperfect hairpins in single strands. Subsequently, these loops or hairpins may be recognized and processed by DNA repair systems. To evaluate the potential role of nucleotide excision repair in repeat instability, we measured the rates of repeat deletion in wild type and excision repair-deficient Escherichia coli strains (using a genetic assay for deletions). The rate of triplet repeat deletion decreased in an E. coli strain deficient in the damage recognition protein UvrA. Moreover, loops containing 23 CTG repeats were less efficiently excised from heteroduplex plasmids after their transformation into the uvrA(-) strain. As a result, an increased proportion of plasmids containing the full-length repeat were recovered after the replication of heteroduplex plasmids containing unrepaired loops. In biochemical experiments, UvrA bound to heteroduplex substrates containing repeat loops of 1, 2, or 17 CAG repeats with a K(d) of about 10-20 nm, which is an affinity about 2 orders of magnitude higher than that of UvrA bound to the control substrates containing (CTG)(n).(CAG)(n) in the linear form. These results suggest that UvrA is involved in triplet repeat instability in cells. Specifically, UvrA may bind to loops formed during replication slippage or in slipped strand DNA and initiate DNA repair events that result in repeat deletion. These results imply a more comprehensive role for UvrA, in addition to the recognition of DNA damage, in maintaining the integrity of the genome.