Pyrimidine dimer excision in Escherichia coli strains deficient in exonucleases V and VII and in the 5'' leads to 3'' exonuclease of DNA polymerase I.

An isogenic series of Escherichia coli strains deficient in various combinations of three 5' leads to 3' exonucleases (exonuclease V, exonuclease VII, and the 5' leads to 3' exonuclease of DNA polymerase I) was constructed and examined for the ability to excise pyrimidine dimers after UV irradiation. Although the recB and recC mutations (deficient in exonuclease V) proved to be incompatible with the polA(Ex) mutation (deficient in the 5' leads to 3' exonuclease of DNA polymerase I), it was possible to reduce the level of the recB,C exonuclease by the use of temperature-sensitive recB270 recC271 mutants. It was found that, by employing strains deficient in exonuclease V, postirradiation DNA degradation could be reduced and dimer excision measurements could be facilitated. Mutants deficient in exonuclease V were found to excise dimers at a rate comparable to that of the wild type. Mutants deficient in exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I are slightly slower than the wild type at removing dimers accumulated after doses in excess of 40 J/m2. However, although strains with reduced levels of exonuclease VII excised dimers at the same rate as the wild type, the addition of an exonuclease VII deficiency to a strain with reduced levels of exonuclease V and the 5' leads to 3' exonuclease of DNA polymerase I caused a marked decrease in the rate and extent of dimer excision. These observations support previous indications that the 5' leads to 3' exonuclease of DNA polymerase I is important in dimer removal and also suggest a role for exonuclease VII in the excision repair process.     

DNA polymerase I-mediated ultraviolet repair synthesis in toluene-treated Escherichia coli.

DNA synthesis after ultraviolet irradiation is low in wild type toluene-treated cells. The level of repair incorporation is greater in strains deficient in DNA polymerase I. The low level of repair synthesis is attributable to the concerted action of DNA polymerase I and polynucleotide ligase. Repair synthesis is stimulated by blocking ligase activity with the addition of nicotinamide mononucleotide (NMN) or the use of a ligase temperature-sensitive mutant. NMN stimulation is specific for DNA polymerase I-mediated repair synthesis, as it is absent in isogenic strains deficient in the polymerase function or the 5' leads to 3' exonuclease function associated with DNA polymerase I. DNA synthesis that is stimulated by NMN is proportional to the ultraviolet exposure at low doses, nonconservative in nature, and is dependent on the uvrA gene product but is independent of the recA gene product. These criteria place this synthesis in the excision repair pathway. The NMN-stimulated repair synthesis requires ATP and is N-ethylmaleimide-resistant. The use of NMN provides a direct means for evaluating the involvement of DNA polymerase I in excision repair.     

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.     

Differential requirement for proliferating cell nuclear antigen in 5' and 3' nick-directed excision in human mismatch repair

Proliferating cell nuclear antigen (PCNA) is involved in mammalian mismatch repair at a step prior to or at mismatch excision, but the molecular mechanism of this process is not fully understood. To examine the role of PCNA in mismatch-provoked and nick-directed excision, orientation-specific mismatch removal of heteroduplexes with a pre-existing nick was monitored in human nuclear extracts supplemented with the PCNA inhibitor protein p21. We show here that, whereas 3' nick-directed mismatch excision was completely inhibited by low concentrations of p21 or a p21 C-terminal fusion protein, 5' nick-directed excision was only partially blocked under the same conditions. No further reduction of the 5' excision was detected when a much higher concentration of p21 C-terminal protein was used. These results suggest the following. (i) There is a differential requirement for PCNA in 3' and 5' nick-directed excision; and (ii) 5' nick-directed excision is conducted by a manner either dependent on or independent of PCNA. Our in vitro reconstitution experiments indeed identified a 5' nick-directed excision pathway that is dependent on PCNA, hMutSalpha, and a partially purified fraction from a HeLa nuclear extract.     

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.     

Host-cell reactivation of alkylated T7 bacteriophage.

Purified T7 phage, treated with methyl methanesulfonate, was assayed on Escherichia coli K-12 host cells deficient in base excision repair. Phage survival, measured immediately after alkylation or following incubation to induce depurination, was lowest on a mutant defective in the polymerase activity of DNA polymerase I (p3478). Strains defective in endonuclease for apurinic sites (AB3027, BW2001) gave a significantly higher level of phage survival, as did the strain defective in the 5'--3' exonuclease activity of DNA polymerase I (RS5065). Highest survival of alkylated T7 phage was observed on the two wild-type strains (AB1157, W3110). These results show that alkylated T7 phage is subject to repair via the base excision repair pathway.     

Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis.

The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylamine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair.     

A defined human system that supports bidirectional mismatch-provoked excision

Mismatch-provoked excision directed by a strand break located 3' or 5' to the mispair has been reconstituted using purified human proteins. While MutSalpha, EXOI, and RPA are sufficient to support hydrolysis directed by a 5' strand break, 3' directed excision also requires MutLalpha, PCNA, and RFC. EXOI interacts with PCNA. RFC and PCNA suppress EXOI-mediated 5' to 3' hydrolysis when the nick that directs excision is located 3' to the mispair and activate 3' to 5' excision, which is dependent on loaded PCNA and apparently mediated by a cryptic EXOI 3' to 5' hydrolytic function. By contrast, RFC and PCNA have only a limited effect on 5' to 3' excision directed by a 5' strand break.     

Self-inflicted wounds, template-directed gap repair and a recombination hotspot. Effects of the mariner transposase

Aberrant repair products of mariner transposition occur at a frequency of approximately 1/500 per target element per generation. Among 100 such mutations in the nonautonomous element peach, most had aberrations in the 5' end of peach (40 alleles), in the 3' end of peach (11 alleles), or a deletion of peach with or without deletion of flanking genomic DNA (29 alleles). Most mariner mutations can be explained by exonuclease "nibble" and host-mediated repair of the double-stranded gap created by the transposase, in contrast to analogous mutations in the P element. In mariner, mutations in the 5' inverted repeat are smaller and more frequent than those in the 3' inverted repeat, but secondary mutations in target elements with a 5' lesion usually had 3' lesions resembling those normally found at the 5' end. We suggest that the mariner transposase distinguishes between the 5' and 3' ends of the element, and that the 5' end is relatively more protected after strand scission. We also find: (1) that homolog-dependent gap repair is a frequent accompaniment to mariner excision, estimated as 30% of all excision events; and (2) that mariner is a hotspot of recombination in Drosophila females, but only in the presence of functional transposase.     

Mismatch repair in human nuclear extracts. Quantitative analyses of excision of nicked circular mismatched DNA substrates,constructed by a new technique employing synthetic oligonucleotides

Mammalian mismatch repair (MMR) systems respond to broad ranges of DNA mismatches and lesions. Kinetic analyses of MMR processing in vitro have focused on base mismatches in a few sequence contexts, because of a lack of general and quantitative MMR assays and because of the difficulty of constructing a multiplicity of MMR substrates, particularly those with DNA lesions. We describe here simple and efficient construction of 11 different MMR substrates, by ligating synthetic oligomers into gapped plasmids generated using sequence-specific N.BstNBI nicking endonuclease, then using sequence-specific nicking endonuclease N.AlwI to introduce single nicks for initiation of 3' to 5' or 5' to 3' excision. To quantitatively assay MMR excision gaps in base-mispaired substrates, generated in human nuclear extracts lacking exogenous dNTPs, we used position- and strand-specific oligomer probes. Mispair-provoked excision along the shorter path from the pre-existing nick toward the mismatch, either 3' to 5' or 5' to 3', predominated over longer path excision by roughly 10:1 to 20:1. MMR excision was complete within 7 min, was highly specific (90:1) for the nicked strand, and was strongly mispair-dependent (at least 40:1). Nonspecific (mismatch-independent) 5' to 3' excision was considerably greater than nonspecific 3' to 5' excision, especially at pre-existing gaps, but was not processive. These techniques can be used to construct and analyze MMR substrates with DNA mismatches or lesions in any sequence context.     

Effects of coordination of diammineplatinum(II) with DNA on the activities of Escherichia coli DNA polymerase I

The effects of the reaction of cis- and trans-diamminedichloroplatinum(II) with DNA have been measured with regard to DNA synthesis, 3'-5' exonuclease (proofreading), and 5'-3' exonuclease (repair) activities of Escherichia coli DNA polymerase I. Both isomers inhibit DNA synthetic activity of the polymerase through an increase in Km values and a decrease in Vmax values for platinated DNA but not for the nucleoside 5'-triphosphates as the varied substrates. The inhibition is a consequence of lowered binding affinity between platinated DNA and DNA polymerase, and of a platination-induced separation of template and primer strands. Strand separation enhances initial rates of 3'-5' excision of [3H]dCMP from platinated DNA (proofreading), while total excision levels of nucleotides are decreased. In contrast to proofreading activity, the 5'-3' exonuclease activity (repair) discriminates between DNA which had reacted with cis- and with trans-diamminedichloroplatinum(II). While both initial rates and total excision are inhibited for the cis isomer, they are almost not affected for the trans isomer. This differential effect could explain why bacterial growth inhibition requires much higher concentrations of trans- than cis-diamminedichloroplatinum(II).     

Mismatch repair in human nuclear extracts. Time courses and ATP requirements for kinetically distinguishable steps leading to tightly controlled 5' to 3' and aphidicolin-sensitive 3' to 5' mispair-provoked excision

Mismatch repair (MMR) systems enhance genomic stability by correcting DNA replication errors. The events in mammalian MMR pathways remain poorly understood. Using HeLa cell nuclear extracts, we analyzed correction of mispairs in circular DNA substrates with single defined nicks and measured excision in the absence of exogenous dNTPs by annealing specific oligonucleotide probes. In reactions initiated by concomitant temperature shift and addition of ATP or Mg(2+) to otherwise complete mixtures on ice, ATP-initiated excision and final error correction lagged behind Mg(2+)-initiated reactions, suggesting a very early requirement for ATP but not its hydrolysis. Subsequent stable commitment (resistance to added excess competitor substrate) began within 30 s, required hydrolyzable ATP, and plateaued after 60-70 s. This may reflect formation of hydrolysis-dependent translocating and/or pre-excision complexes. Excision along shorter nick-mispair paths began 15 s later than commitment. Both 3' to 5' and 5' to 3' excision gaps appeared at rates of approximately 0.0055 of final yields per second, respectively, 30 or 2.5 times the nonspecific excision rates. The lag between 3' to 5' excision gaps at two different positions yielded an excision progress rate of 5.2 nucleotides/s. In both substrates, corrected products appeared at fractional rates of 0.0027 of final yield per second. Aphidicolin, known to inhibit both the DNA synthesis and 3' to 5' exonuclease activities of polymerases delta and epsilon, reduced appearance of 3' to 5' excision tracts roughly 4-fold at 90 microm but had no effect on 5' to 3' excision.     

The phosphonomethyl analogue of 3-phosphoglycerate is a potent competitive inhibitor of phosphoglycerate mutases.

Deoxyribonuclease IV, a 5'-3' exonuclease degrading double-stranded DNA from intra-strand nicks, has been purified from the chromatin of rat liver cells. The enzyme, which has an Mr of 58000, excises the apurinic (AP) sites from a depurinated DNA nicked 5' to these AP sites with the chromatin AP endonuclease. The excision is not the result of hydrolysis of the phosphodiester bond 3' to the AP sites since the excision product does not behave as deoxyribose 5-phosphate but as its 2,3-unsaturated derivative. This result suggests that, to remove the AP sites from the DNA nicked by an AP endonuclease, the chromatin deoxyribonuclease IV rather acts as a catalyst of beta-elimination.     

Deoxyribonucleic acid repair in Escherichia coli mutants deficient in the 5''----3'' exonuclease activity of deoxyribonucleic acid polymerase I and exonuclease VII.

A series of Escherichia coli strains deficient in the 5'----3' exonuclease activity associated with deoxyribonucleic acid (DNA) polymerase I (exonuclease VI) and exonuclease VII has been constructed. Both of these enzymes are capable of pyrimidine dimer excision in vitro. These strains were examined for conditional lethality, sensitivity to ultraviolet (UV) and X-irradiation, postirradiation DNA degradation, and ability to excise pyrimidine dimers. It was found that strains deficient in both exonuclease VI (polAex-) and exonuclease VII (xseA-) are significantly reduced in their ability to survive incubation at elevated temperature (43 degrees C) beyond the reduction previously observed for the polAex single mutants. The UV and X-ray sensitivity of the exonuclease VI-deficient strains was not increased by the addition of the xseA7 mutation. Mutants deficient in both enzymes are about as efficient as wild-type strains at excising dimers produced by up to 40 J/m2 UV. At higher doses strains containing only polAex- mutations show reduced ability to excise dimers; however, the interpretation of dimer excision data at these doses is complicated by extreme postirradiation DNA degradation in these strains. The additional deficiency in the polAex xseA7 double-mutant strains has no significant effect on either postirradiation DNA degradation or the apparent deficiency in dimer excision at high UV doses observed in polAex single mutants.     

Human exonuclease I is required for 5' and 3' mismatch repair.

We have partially purified a human activity that restores mismatch-dependent, bi-directional excision to a human nuclear extract fraction depleted for one or more mismatch repair excision activities. Human EXOI co-purifies with the excision activity, and the purified activity can be replaced by near homogeneous recombinant hEXOI. Despite the reported 5' to 3' hydrolytic polarity of this activity, hEXOI participates in mismatch-provoked excision directed by a strand break located either 5' or 3' to the mispair. When the strand break that directs repair is located 3' to the mispair, hEXOI- and mismatch-dependent gap formation in excision-depleted extracts requires both hMutSalpha and hMutLalpha. However, excision directed by a 5' strand break requires hMutSalpha but can occur in absence of hMutLalpha. In systems comprised of pure components, the 5' to 3' hydrolytic activity of hEXOI is activated by hMutSalpha in a mismatch-dependent manner. These observations indicate a hydrolytic function for hEXOI in 5'-heteroduplex correction. The involvement of hEXOI in 3'-heteroduplex repair suggests that it has a regulatory/structural role in assembly of the 3'-excision complex or that the protein possesses a cryptic 3' to 5' hydrolytic activity.     

Carcinogen-induced DNA repair in nucleotide-permeable Escherichia coli cells. Induction of DNA repair by the carcinogens methyl and ethyl nitrosourea and methyl methanesulfonate.

Ether-permeabilized (nucleotide-permeable) cells of Escherichia coli show excision repair of their DNA after having been exposed to the carcinogens N-methyl-N-nitrosourea (MeNOUr), N-ethyl-N-nitrosourea (EtNOUr) and methyl methanesulfonate (MeSO2OMe) which are known to bind covalently to DNA. Defect mutations in genes uvrA, uvrB, uvrC, recA, recB, recC and rep did not inhibit this excision repair. Enzymic activities involved in this repair were identified by measuring size reduction of DNA, DNA degradation to acid-soluble nucleotides and repair polymerization. 1. In permeabilized cells methyl and ethyl nitrosourea induced endonucleolytic cleavage of endogenous DNA, as determined by size reduction of denatured DNA in neutral and alkaline sucrose gradients. An enzymic activity from E. coli K-12 cell extracts was purified (greater than 2000-fold) and was found to cleave preferentially methyl-nitrosourea-treated DNA and to convert the methylated supercoiled DNA duplex (RFI) of phage phiX 174 into the nicked circular form. 2. Degradation of alkylated cellular DNA to acid solubility was diminished in a mutant lacking the 5' leads to 3' exonucleolytic activity of DNA polymerase I but was not affected in a mutant which lacked the DNA polymerizing but retained the 5' leads 3' exonucleolytic activity of DNA polymerase I. 3. An easily measurable effect is carcinogen-induced repair polymerization, making it suitable for detection of covalent binding of carcinogens and potentially carcinogenic compounds.