Multiple pathways for repair of hydrogen peroxide-induced DNA damage in Escherichia coli.

The repair response of Escherichia coli to hydrogen peroxide has been examined in mutants which show increased sensitivity to this agent. Four mutants were found to show increased in vivo sensitivity to hydrogen peroxide compared with wild type. These mutants, in order of increasing sensitivity, were recA, polC, xthA, and polA. The polA mutants were the most sensitive, implying that DNA polymerase I is required for any repair of hydrogen peroxide damage. Measurement of repair synthesis after hydrogen peroxide treatment demonstrated normal levels for recA mutants, a small amount for xthA mutants, and none for polA mutants. This is consistent with exonuclease III being required for part of the repair synthesis seen, while DNA polymerase I is strictly required for all repair synthesis. Sedimentation analysis of cellular DNA after hydrogen peroxide treatment showed that reformation was absent in xthA, polA, and polC(Ts) strains but normal in a recA cell line. By use of a lambda phage carrying a recA-lacZ fusion, we found hydrogen peroxide does not induce the recA promoter. Our findings indicate two pathways of repair for hydrogen peroxide-induced DNA damage. One of these pathways would utilize exonuclease III, DNA polymerase III, and DNA polymerase I, while the other would be DNA polymerase I dependent. The RecA protein seems to have little or no direct function in either repair pathway.     

Isolation of cDNA clones encoding a human apurinic/apyrimidinic endonuclease that corrects DNA repair and mutagenesis defects in E. coli xth (exonuclease III) mutants.

Apurinic/apyrimidinic (AP) sites in cellular DNA are considered to be both cytotoxic and mutagenic, and can arise spontaneously or following exposure to DNA damaging agents. We have isolated cDNA clones which encode an endonuclease, designated HAP1 (human AP endonuclease 1), that catalyses the initial step in AP site repair in human cells. The predicted HAP1 protein has an Mr of 35,500 and shows striking sequence similarity (93% identity) to BAP 1, a bovine AP endonuclease enzyme. Significant sequence homology to two bacterial DNA repair enzymes, E. coli exonuclease III and S. pneumoniae ExoA proteins, and to Drosophila Rrp1 protein is also apparent. We have expressed the HAP1 cDNA in E. coli mutants lacking exonuclease III (xth), endonuclease IV (nfo), or both AP endonucleases. The HAP1 protein can substitute for exonuclease III, but not for endonuclease IV, in respect of some, but not all, DNA repair and mutagenesis functions. Moreover, a dut xth (ts) double mutant, which is nonviable at 42 degrees C due to an accumulation of unrepaired AP sites following excision of uracil from DNA, was rescued by expression of the HAP1 cDNA. These results indicate that AP endonucleases show remarkable conservation of both primary sequence and function. We would predict that the HAP1 protein is important in human cells for protection against the toxic and mutagenic effects of DNA damaging agents.     

Drosophila Rrp1 complements E. coli xth nfo mutants: protection against both oxidative and alkylation-induced DNA damage.

Drosophila Rrp1 protein has four tightly associated enzymatic activities: DNA strand transfer, ssDNA renaturation, dsDNA 3'-exonuclease and apurinic/apyrimidinic (AP) endonuclease. The carboxy-terminal region of Rrp1 is homologous to Escherichia coli exonuclease III and several eukaryotic AP endonucleases. All members of this protein family cleave abasic sites. Rrp1 protein was expressed under the control of the E. coli RNA polymerase tac promoter (pRrp1-tac) in two repair deficient E. coli strains (BW528 and LG101) lacking both exonuclease III (xth) and endonuclease IV (nfo). Rrp1 confers resistance to killing by oxidative, antitumor and alkylating agents that damage DNA (hydrogen peroxide, t-butylhydroperoxide, bleomycin, methyl methanesulfonate, and mitomycin C). Complementation of the repair deficiency by Rrp1 provides up to a two log increase in survival and requires the C-terminal nuclease region of Rrp1, but not its N-terminal region. The AP endonuclease activity in extracts from the repair deficient strain LG101 is increased up to 12-fold when the strain contains pRrp1-tac. These results indicate that pRrp1-tac directs the synthesis of active enzyme, and that the nuclease activities of Rrp1 are likely to be the cause of the increased resistance to DNA damage of the mutant cells.     

Multiple pathways for repair of oxidative DNA damages caused by X rays and hydrogen peroxide in Escherichia coli.

The responses of Escherichia coli to X rays and hydrogen peroxide were examined in mutants which are deficient in one or more DNA repair genes. Mutant cells deficient in either exonuclease III (xthA) or endonuclease IV (nfo) had normal resistance to X rays, but an xthA-nfo double mutant showed a sensitivity increased over that of either parental strain. A DNA polymerase I mutant (polA) was more sensitive than the xthA-nfo mutant. Cells bearing mutations in all of the polA, xthA, and nfo genes were more sensitive to X rays than polA and xthA-nfo mutants. Similar repair responses were obtained by exposing these mutant cells to hydrogen peroxide, with the exception of the xthA mutant, which was hypersensitive to this agent. The DNA polymerase III mutant (polC(Ts)) was slightly more sensitive to the agents than the wild-type strain at the restrictive temperature. The sensitivity of the polC-xthA-nfo mutant to X rays and hydrogen peroxide was greater than that of polC but almost the same as that of the xthA-nfo mutant. From these results it appears that there are at least four repair pathways, the DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase I-, exonuclease III/endonuclease IV and DNA polymerase III-, and exonuclease III/endonuclease IV-dependent pathways, for the repair of oxidative DNA damages in E. coli.     

Repair of DNA lesions induced by hydrogen peroxide in the presence of iron chelators in Escherichia coli: participation of endonuclease IV and Fpg

In Escherichia coli, the repair of lethal DNA damage induced by H(2)O(2) requires exonuclease III, the xthA gene product. Here, we report that both endonuclease IV (the nfo gene product) and exonuclease III can mediate the repair of lesions induced by H(2)O(2) under low-iron conditions. Neither the xthA nor the nfo mutants was sensitive to H(2)O(2) in the presence of iron chelators, while the xthA nfo double mutant was significantly sensitive to this treatment, suggesting that both exonuclease III and endonuclease IV can mediate the repair of DNA lesions formed under such conditions. Sedimentation studies in alkaline sucrose gradients also demonstrated that both xthA and nfo mutants, but not the xthA nfo double mutant, can carry out complete repair of DNA strand breaks and alkali-labile bonds generated by H(2)O(2) under low-iron conditions. We also found indications that the formation of substrates for exonuclease III and endonuclease IV is mediated by the Fpg DNA glycosylase, as suggested by experiments in which the fpg mutation increased the level of cell survival, as well as repair of DNA strand breaks, in an AP endonuclease-null background.     

Excision repair of thymine glycols, urea residues, and apurinic sites in Escherichia coli

The genetic requirements for the excision repair of thymine glycols, urea residues, and apurinic (AP) sites were examined by measuring the survival in Escherichia coli mutants of phi X174 replicative form (RF) I transfecting DNA containing selectively introduced lesions. phi X RF I DNA containing thymine glycols was inactivated at a greater rate in mutants deficient in endonuclease III (nth) than in wild-type hosts, suggesting that endonuclease III is involved in the repair of thymine glycols in vivo. phi X RF I DNA containing thymine glycols was also inactivated at a greater rate in mutants that were deficient in both exonuclease III and endonuclease IV (xth nfo) than in wild-type hosts, suggesting that a class II AP endonuclease is required for the in vivo processing of thymine glycols. phi X duplex-transfecting DNA containing urea residues or AP sites was inactivated at a greater rate in xth nfo double mutants than in wild-type, but not single-mutant, hosts, suggesting that exonuclease III or endonuclease IV is required for the repair of these damages and that either activity can substitute for the other. These data are in agreement with the known in vitro substrate specificities of endonuclease III, exonuclease III, and endonuclease IV.     

Repair of x-ray-induced deoxyribonucleic acid single-strand breaks in xth mutants of Escherichia coli.

An exonuclease III-deficient strain of Escherichia coli K-12, BW2001 (xthA11), was unable to perform rapid repair of X-ray-induced deoxyribonucleic acid single-strand breaks and appeared to have a defect in the priming of the 3'-termini necessary for initiation of repair synthesis at the breaks. This defect cannot be explained solely by the lack of exonuclease III activity, because other xth mutants tested, including a deletion mutant, repaired radiation-induced strand breaks at close to the normal rate.     

Normal processing of AP sites in Apn1-deficient Saccharomyces cerevisiae is restored by Escherichia coli genes expressing either exonuclease III or endonuclease III

Escherichia coli exonuclease III and endonuclease III are two distinct DNA-repair enzymes that can cleave apurinic/apyrimidinic (AP) sites by different mechanisms. While the AP endonuclease activity of exonuclease III generates a 3'-hydroxyl group at AP sites, the AP lyase activity of endonuclease III produces a 3'-α,β unsaturated aldehyde that prevents DNA-repair synthesis. Saccharomyces cerevisiae Apn1 is the major AP endonuclease/3'-diesterase that also produces a 3'-hydroxyl group at the AP site, but it is unrelated to either exonuclease III or endonuclease III. apn1 deletion mutants are unable to repair AP sites generated by the alkylating agent methyl methane sulphonate and display a spontaneous mutator phenotype. This work shows that either exonuclease III or endonuclease III can functionally replace yeast Apn1 in the repair of AP sites. Two conclusions can be derived from these findings. The first of these conclusions is that yeast cells can complete the repair of AP sites even though they are cleaved by AP lyase. This implies that AP lyase can contribute significantly to the repair of AP sites and that yeast cells have the ability to process the α,β unsaturated aldehyde produced by endonuclease III. The second of these conclusions is that unrepaired AP sites are strictly the cause of the high spontaneous mutation rate in the apn1 deletion mutant.     

Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1).

Two forms of DNA base excision-repair (BER) have been observed: a 'short-patch' BER pathway involving replacement of one nucleotide and a 'long-patch' BER pathway with gap-filling of several nucleotides. The latter mode of repair has been investigated using human cell-free extracts or purified proteins. Correction of a regular abasic site in DNA mainly involves incorporation of a single nucleotide, whereas repair patches of two to six nucleotides in length were found after repair of a reduced or oxidized abasic site. Human AP endonuclease, DNA polymerase beta and a DNA ligase (either III or I) were sufficient for the repair of a regular AP site. In contrast, the structure-specific nuclease DNase IV (FEN1) was essential for repair of a reduced AP site, which occurred through the long-patch BER pathway. DNase IV was required for cleavage of a reaction intermediate generated by template strand displacement during gap-filling. XPG, a related nuclease, could not substitute for DNase IV. The long-patch BER pathway was largely dependent on DNA polymerase beta in cell extracts, but the reaction could be reconstituted with either DNA polymerase beta or delta. Efficient repair of gamma-ray-induced oxidized AP sites in plasmid DNA also required DNase IV. PCNA could promote the Pol beta-dependent long-patch pathway by stimulation of DNase IV.     

Serratia marcescens rpr gene sensitizes Escherichia coli wild-type, xth, and nfo strains to methyl methanesulphonate

It is reported here that the rpr DNA repair gene of Serratia marcescens does not complement an Escherichia coli xth nfo AP endonuclease mutation for resistance to methyl methanesulphonate (MMS). Rather, rpr sensitized Escherichia coli wild-type, xth, and nfo strains to MMS. Also, it was found that rpr could not complement a triple tag alkA recA mutation in E. coli, indicating that there are limits to rpr complementing capabilities. It was determined that rpr gene dosage was not a factor in recA complementation. MMS sensitization of an E. coli wild-type strain, however, was directly related to rpr copy number. These data indicate that Rpr does not have an associated AP endonuclease activity, and that it is incapable of substituting for Tag I, Tag II, and RecA in a tag alkA recA background.     

Single‐nucleotide and long‐patch base excision repair of DNA damage in plants

Base excision repair (BER) is a critical pathway in cellular defense against endogenous or exogenous DNA damage. This elaborate multistep process is initiated by DNA glycosylases that excise the damaged base, and continues through the concerted action of additional proteins that finally restore DNA to the unmodified state. BER has been subject to detailed biochemical analysis in bacteria, yeast and animals, mainly through in vitro reproduction of the entire repair reaction in cell‐free extracts. However, an understanding of this repair pathway in plants has consistently lagged behind. We report the extension of BER biochemical analysis to plants, using Arabidopsis cell extracts to monitor repair of DNA base damage in vitro. We have used this system to demonstrate that Arabidopsis cell extracts contain the enzymatic machinery required to completely repair ubiquitous DNA lesions, such as uracil and abasic (AP) sites. Our results reveal that AP sites generated after uracil excision are processed both by AP endonucleases and AP lyases, generating either 5′‐ or 3′‐blocked ends, respectively. We have also found that gap filling and ligation may proceed either through insertion of just one nucleotide (short‐patch BER) or several nucleotides (long‐patch BER). This experimental system should prove useful in the biochemical and genetic dissection of BER in plants, and contribute to provide a broader picture of the evolution and biological relevance of DNA repair pathways.     

2'-deoxyribonolactone lesion produces G->A transitions in Escherichia coli

2'-deoxyribonolactone (dL) is a C1'-oxidized abasic site damage generated by a radical attack on DNA. Numerous genotoxic agents have been shown to produce dL including UV and gamma-irradiation, ene-dye antibiotics etc. At present the biological consequences of dL present in DNA have been poorly documented, mainly due to the lack of method for introducing the lesion in oligonucleotides. We have recently designed a synthesis of dL which allowed investigation of the mutagenicity of dL in Escherichia coli by using a genetic reversion assay. The lesion was site-specifically incorporated in a double-stranded bacteriophage vector M13G*1, which detects single-base-pair substitutions at position 141 of the lacZalpha gene by a change in plaque color. In E.coli JM105 the dL-induced reversion frequency was 4.7 x 10(-5), similar to that of the classic abasic site 2'-deoxyribose (dR). Here we report that a dL residue in a duplex DNA codes mainly for thymidine. The processing of dL in vivo was investigated by measuring lesion-induced mutation frequencies in DNA repair deficient E.coli strains. We showed a 32-fold increase in dL-induced reversion rate in AP endonuclease deficient (xth nfo) mutant compared with wild-type strain, indicating that the Xth and Nfo AP endonucleases participate in dL repair in vivo.     

Endonuclease IV (nfo) mutant of Escherichia coli.

A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested.     

Processing in vitro of an abasic site reacted with methoxyamine: a new assay for the detection of abasic sites formed in vivo.

In this study we demonstrate that the different substrate recognition properties of bacterial and human AP endonucleases might be used to quantify and localize apurinic (AP) sites formed in DNA in vivo. By using a model oligonucleotide containing a single AP site modified with methoxyamine (MX), we show that endonuclease III and IV of E. coli are able to cleave the alkoxyamine-adducted site whereas a partially purified HeLa AP endonuclease and crude cell-free extracts from HeLa cells are inhibited by this modification. In addition MX-modified AP sites in a DNA template retain their ability to block DNA synthesis in vitro. Since MX can efficiently react with AP sites formed in mammalian cells in vivo we propose that the MX modified abasic sites thus formed can be quantitated and localized at the level of the individual gene by subsequent site specific cleavage by either E. coli endonuclease III or IV in vitro.     

Antimutator role of the DNA glycosylase mutY gene in Helicobacter pylori

Helicobacter pylori has a highly variable genome with ongoing diversification via inter- and intragenomic recombination and spontaneous mutation. DNA repair genes modulating mutation and recombination rates that influence diversification have not been well characterized for H. pylori. To examine the role of putative base excision repair ung and mutY glycosylase and xthA apurinic/apyrimidinic endonuclease genes in H. pylori, mutants of each were constructed in strain JP26 by allelic exchange. Spontaneous mutation frequencies of JP26 mutY mutants, assessed by rifampin resistance, were consistently higher (26-fold) than that of the wild type, whereas the ung and xthA mutants showed smaller increases. In trans complementation of the JP26 mutY mutant restored spontaneous mutation frequencies to wild-type levels. In cross-species studies, H. pylori mutY complemented an Escherichia coli mutY mutant and vice versa. In contrast, the ung and mutY mutants did not show higher frequencies of intergenomic recombination or greater sensitivity to UV-induced DNA damage than the wild type. The H. pylori mutY open reading frame contains an eight-adenine homonucleotide tract; we provide evidence that this is subject to slipped-strand mispairing, leading to frameshifts that eliminate gene function. Our findings indicate that H. pylori possesses phase-variable base excision repair, consistent with a tension between repair and mutation.     

Chloroplast lysates support directed mutagenesis via modified DNA and chimeric RNA/DNA oligonucleotides

Chimeric RNA/DNA and modified DNA oligonucleotides have been shown to direct gene-conversion events in vitro through a process involving proteins from several DNA-repair pathways. Recent experiments have extended the utility of these molecules to plants, and we previously demonstrated that plant cell-free extracts are competent to support oligonucleotide-directed genetic repair. Using this system, we are studying Arabidopsis DNA-repair mutants and the role of plant proteins in the DNA-repair process. Here we describe a method for investigating mechanisms of plastid DNA-repair pathways. Using a genetic readout system in bacteria and chimeric or modified DNA oligonucleotides designed to direct the conversion of mutations in antibiotic resistance genes, we have developed an assay for genetic repair of mutations in a spinach chloroplast lysate system. We report genetic repair of point and frameshift mutations directed by both types of modified oligonucleotides. This system enables the mechanistic study of plastid gene repair and facilitates the direct comparison between plant nuclear and organelle DNA-repair pathways.     

Genetic and Biochemical Characterization of Human AP Endonuclease 1 Mutants Deficient in Nucleotide Incision Repair Activity

Background

Human apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair enzyme involved in both base excision repair (BER) and nucleotide incision repair (NIR) pathways. In the BER pathway, APE1 cleaves DNA at AP sites and 3′-blocking moieties generated by DNA glycosylases. In the NIR pathway, APE1 incises DNA 5′ to a number of oxidatively damaged bases. At present, physiological relevance of the NIR pathway is fairly well established in E. coli, but has yet to be elucidated in human cells.

Methodology/Principal Finding

We identified amino acid residues in the APE1 protein that affect its function in either the BER or NIR pathway. Biochemical characterization of APE1 carrying single K98A, R185A, D308A and double K98A/R185A amino acid substitutions revealed that all mutants exhibited greatly reduced NIR and 3′→5′ exonuclease activities, but were capable of performing BER functions to some extent. Expression of the APE1 mutants deficient in the NIR and exonuclease activities reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to an alkylating agent, methylmethanesulfonate, suggesting that our APE1 mutants are able to repair AP sites. Finally, the human NIR pathway was fully reconstituted in vitro using the purified APE1, human flap endonuclease 1, DNA polymerase β and DNA ligase I proteins, thus establishing the minimal set of proteins required for a functional NIR pathway in human cells.

Conclusion/Significance

Taken together, these data further substantiate the role of NIR as a distinct and separable function of APE1 that is essential for processing of potentially lethal oxidative DNA lesions.     

Repair of tandem base lesions in DNA by human cell extracts generates persisting single-strand breaks

Clustered DNA damage, where two or more lesions are located proximal to each other on the same or opposite DNA strands, is frequently produced as a result of exposure to ionising radiation. It has been suggested that such complex damaged sites pose problems for repair pathways. In this study, we addressed the question of how two 8-oxoguanine lesions, located two nucleotides apart on the same DNA strand, are repaired. We find that in human cell extracts repair of either of the 8-oxoguanine lesions within a tandem damaged site is initiated randomly and that the majority of the initiated repair proceeds to completion. However, a fraction of the initiated repair is delayed at the stage of an incised AP site and the rate of further processing of this incised AP site is dependent on the position of the remaining 8-oxoguanine. If the remaining 8-oxoguanine residue is located near the 5' terminus of the incised abasic site, repair continues as efficiently as repair of a single 8-oxoguanine residue. However, repair is delayed after the incision step when the remaining 8-oxoguanine residue is located near the 3' terminus. Although the presence of the 8-oxoguanine residue near the 3' terminus did not affect either DNA polymerase beta activity or poly(ADP)ribose polymerase-1 affinity and turnover on an incised AP site, we find that 8-oxoguanine-DNA glycosylase has reduced ability to remove an 8-oxoguanine residue located near the 3' terminus of the incised AP site. We find that binding of the 8-oxoguanine-DNA glycosylase to this 8-oxoguanine residue inhibits DNA repair synthesis by DNA polymerase beta, thus delaying repair. We propose that interference between a DNA glycosylase and DNA polymerase during the repair of tandem lesions may lead to accumulation of the intermediate products that contain persisting DNA strand breaks.     

Deoxyribonucleic acid repair in vitro by extracts of Escherichia coli.

Deoxyribonucleic acid (DNA) from bacteriophage T7 has been used to monitor the capacity of gently lysed extracts of Escherichia coli to perform repair resynthesis after ultraviolet (UV) irradiation. Purified DNA damaged by up to 100 J of UV radiation per m2 was treated with an endonuclease from Micrococcus luteus that introduces single-strand breaks in irradiated DNA. This DNA was then used as a substrate to study repair resynthesis by extracts of E. coli. It was found that incubation with the extract and exogenous nucleoside triphosphates under suitable assay conditions resulted in removal of all pyrimidine dimers and restoration of the substrate DNA to its original molecular weight. Repair resynthesis, detected as nonconservative, UV-stimulated DNA synthesis, was directly proportional tothe number of pyrimidine dimers introduced by radiation. The repair mode described here appears to require DNA polymerase I since it does no occur at the restrictive temperature in polA12 mutants, which contain a thermolabile polymerase. The addition of purified DNA polymerase I to extracts made from a polA mutant restores the ability to complete repair at the restrictive temperature.     

Role of the Nfo and ExoA apurinic/apyrimidinic endonucleases in radiation resistance and radiation-induced mutagenesis of Bacillus subtilis spores

The roles of DNA repair by apurinic/apyrimidinic (AP) endonucleases alone, and together with DNA protection by α/β-type small acid-soluble spore proteins (SASP), in Bacillus subtilis spore resistance to different types of radiation have been studied. Spores lacking both AP endonucleases (Nfo and ExoA) and major SASP were significantly more sensitive to 254-nm UV-C, environmental UV (>280 nm), X-ray exposure, and high-energy charged (HZE)-particle bombardment and had elevated mutation frequencies compared to those of wild-type spores and spores lacking only one or both AP endonucleases or major SASP. These findings further implicate AP endonucleases and α/β-type SASP in repair and protection, respectively, of spore DNA against effects of UV and ionizing radiation.