Complementation of the xeroderma pigmentosum DNA repair synthesis defect with Escherichia coli UvrABC proteins in a cell-free system.

A newly developed cell-free system was used to study DNA repair synthesis carried out by extracts from human cell lines in vitro. Extracts from a normal human lymphoid cell line and from cell lines established from individuals with hereditary dysplastic nevus syndrome perform damage-dependent repair synthesis in plasmid DNA treated with cis- or trans-diamminedichloro-platinum(II) or irradiated with ultraviolet light. Cell extracts of xeroderma pigmentosum origin (complementation groups A, C, D, and G) are deficient in DNA repair synthesis. When damaged plasmid DNA was pretreated with purified Escherichia coli UvrABC proteins, xeroderma pigmentosum cell extracts were able to carry out DNA repair synthesis. The ability of E. coli UvrABC proteins to complement xeroderma pigmentosum cell extracts indicates that the extracts are deficient in incision, but can carry out later steps of repair. Thus the in vitro system provides results that are in agreement with the incision defect found from studies of xeroderma pigmentosum cells.     

Interstrand Cross-Links Induce DNA Synthesis in Damaged and Undamaged Plasmids in Mammalian Cell Extracts

Mammalian cell extracts have been shown to carry out damage-specific DNA repair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-link induces DNA synthesis in both the damaged plasmid and a second homologous unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly stimulates repair synthesis in the cross-linked plasmid. Heterologous DNAs also stimulate repair synthesis to variable extents. Psoralen monoadducts and double-strand breaks do not induce repair synthesis in the unmodified plasmid, indicating that such incorporation is specific to interstrand cross-links. This induced repair synthesis is consistent with previous evidence indicating a recombinational mode of repair for interstrand cross-links. DNA synthesis is compromised in extracts from mutants (deficient in ERCC1, XPF, XRCC2, and XRCC3) which are all sensitive to DNA cross-linking agents but is normal in extracts from mutants (XP-A, XP-C, and XP-G) which are much less sensitive. Extracts from Fanconi anemia cells exhibit an intermediate to wild-type level of activity dependent upon the complementation group. The DNA synthesis deficit in ERCC1- and XPF-deficient extracts is restored by addition of purified ERCC1-XPF heterodimer. This system provides a biochemical assay for investigating mechanisms of interstrand cross-link repair and should also facilitate the identification and functional characterization of cellular proteins involved in repair of these lesions.     

Repair synthesis by human cell extracts in cisplatin-damaged DNA is preferentially determined by minor adducts.

During reaction of cis-diamminedichloroplatinum(II) (cis-DDP) with DNA, a number of adducts are formed which may be discriminated by the excision-repair system. An in vitro excision-repair assay with human cell-free extracts has been used to assess the relative repair extent of monofunctional adducts, intrastrand and interstrand cross-links of cis-DDP on plasmid DNA. Preferential removal of cis-DDP 1,2-intrastrand diadducts occurred in the presence of cyanide ions. In conditions where cyanide treatment removed 85% of total platinum adducts while approximately 70% of interstrand cross-links remained in plasmid DNA, no significant variation in repair synthesis by human cell extracts was observed. Then, we constructed three types of plasmid DNA substrates containing mainly either monoadducts, 1,2-intrastrand cross-links or interstrand cross-links lesions. The three plasmid species were modified in order to obtain the same extent of total platinum DNA adducts per plasmid. No DNA repair synthesis was detected with monofunctional adducts during incubation with human whole cell extracts. However, a two-fold increase in repair synthesis was found when the proportion of interstrand cross-links in plasmid DNA was increased by 2-3 fold. These findings suggest that (i) cis-DDP 1,2-intrastrand diadducts are poorly repaired by human cell extracts in vitro, (ii) among other minor lesions potentially cyanide-resistant, cis-DDP interstrand cross-links represent a major lesion contributing to the repair synthesis signal in the in vitro assay. These results could account for the drug efficiency in vivo.     

DNA repair replication by soluble extracts from human lymphoid cell lines

A system is described in which extracts from human cells can perform repair replication on DNA damaged by ultraviolet light or chemical carcinogens. Whole cell extracts from lymphoid cell lines are incubated with damaged plasmid DNA circles at 30 degrees C in the presence of ATP and the four deoxynucleoside triphosphates. Repair synthesis is monitored by the incorporation of alpha-32P-dATP into closed circular plasmid molecules. Analysis of the time course of the reaction suggests that the slowest step in repair is incision, rather than polymerization or ligation. The size of repair patches inserted into ultraviolet-irradiated DNA during a reaction was estimated by substitution of thymidine triphosphate with 5-bromodeoxyuridine triphosphate and sedimentation in alkaline cesium chloride gradients. Patches with heterogeneous sizes of less than 120 bases were observed.     

Heteroduplex repair in extracts of human HeLa cells

A general repair process for DNA heteroduplexes has been detected in HeLa cell extracts. Using a variety of M13mp2 DNA substrates containing single-base mismatches and extra nucleotides, extensive repair is observed after incubation with HeLa cell cytoplasmic extracts and subsequent transfection of bacterial cells with the treated DNA. Most, but not all, mispairs as well as two frameshift heteroduplexes are repaired efficiently. Parallel measurements of repair in HeLa extracts and in Escherichia coli suggest that repair specificities are similar for the two systems. The presence of a nick in the molecule is required for efficient repair in HeLa cell extracts, and the strand containing the nick is the predominantly repaired strand. Mismatch-dependent DNA synthesis is observed when radiolabeled restriction fragments, produced by reaction of the extract with heteroduplex and homoduplex molecules, are compared. Specific labeling of fragments, representing a region of approximately 1,000 base pairs and containing the nick and the mismatch, is detected for the heteroduplex substrate but not the homoduplex. The repair reaction is complete after 20 min and requires added Mg2+, ATP, and an ATP-regenerating system, but not dNTPs, which are present at sufficient levels in the extract. An inhibitor of DNA polymerase beta, dideoxythimidine 5'-triphosphate, does not inhibit mismatch-specific DNA synthesis. Aphidicolin, an inhibitor of DNA polymerases alpha, delta, and epsilom, inhibits both semiconservative replication and repair synthesis in the extract. Butylphenyl-dGTP also inhibits both replicative and repair synthesis but at a concentration known to inhibit DNA polymerase alpha preferentially rather than delta or epsilon. This suggests that DNA polymerase alpha may function in mismatch repair.     

Repair synthesis by human cell extracts in DNA damaged by cis- and trans-diamminedichloroplatinum(II)

DNA damage was induced in closed circular plasmid DNA by treatment with cis- or trans-diamminedichloroplatinum(II). These plasmids were used as substrates in reactions to give quantitative measurements of DNA repair synthesis mediated by cell free extracts from human lymphoid cell lines. Adducts induced by both drugs stimulated repair synthesis in a dose dependent manner by an ATP-requiring process. Measurements by an isopycnic gradient sedimentation method gave an upper limit for the average patch sizes in this in vitro system of around 140 nucleotides. It was estimated that up to 3% of the drug adducts induce the synthesis of a repair patch. The repair synthesis is due to repair of a small fraction of frequent drug adducts, rather than extensive repair of a rare subclass of lesions. Nonspecific DNA synthesis in undamaged plasmids, caused by exonucleolytic degradation and resynthesis, was reduced by repeated purification of intact circular forms. An extract made from cells belonging to xeroderma pigmentosum complementation group A was deficient in repair synthesis in response to the presence of cis- or trans-diamminedichloroplatinum(II) adducts in DNA.     

Repair kinetics of trans-4-hydroxynonenal-induced cyclic 1,N2-propanodeoxyguanine DNA adducts by human cell nuclear extracts

trans-4-Hydroxynonenal (HNE) is a major peroxidation product of omega-6 polyunsaturated fatty acids. The reaction of HNE with DNA produces four diastereomeric 1,N(2)-gamma-hydroxypropano adducts of deoxyguanosine (HNE-dG); background levels of these adducts have been detected in tissues of animals and humans. There is evidence to suggest that these adducts are mutagenic and involved in liver carcinogenesis in patients with Wilson's disease and in other human cancers. Here, we present biochemical evidence that in human cell nuclear extracts the HNE-dG adducts are repaired by the nucleotide excision repair (NER) pathway. To investigate the recognition and repair of HNE-dG adducts in human cell extracts, we prepared plasmid DNA substrates modified by HNE. [(32)P]-Postlabeling/HPLC determined that the HNE-dG adduct levels were approximately 1200/10(6) dG of plasmid DNA substrate. We used this substrate in an in vitro repair-synthesis assay to study the complete repair of HNE-induced DNA adducts in cell-free extracts. We observed that nuclear extracts from HeLa cells incorporated a significant amount of alpha[(32)P]dCTP in DNA that contained HNE-dG adducts by comparison with UV-irradiated DNA as the positive control. Such repair synthesis for UV damage or HNE-dG adducts did not occur in XPA cell nuclear extracts that lack the capacity for NER. However, XPA cells complemented with XPA protein restored repair synthesis for both of these adducts. To verify that HNE-dG adducts in DNA were indeed repaired, we measured HNE-dG adducts in the post-repaired DNA substrates by the [(32)P]-postlabeling/HPLC method, showing that 50-60% of HNE-dG adducts were removed from the HeLa cell nuclear extracts after 3 h at 30 degrees C. The repair kinetics indicated that the excision rate is faster than the rate of gap-filling/DNA synthesis. Furthermore, the HNE-dG adduct isomers 2 and 4 appeared to be repaired more efficiently at early time points than isomers 1 and 3.     

Deoxyribonucleic acid synthesis in isolated chloroplasts and chloroplast extracts of maize

Isolated chloroplasts are capable of synthesizing chloroplast DNA in the presence of Mg2+ and deoxynucleoside triphosphates. The in vitro reaction proceeds for at least 60 min and is inhibited by KC1 and N-ethylmaleimide. Stretches of several hundred nucleotides in length are synthesized within an hour. Little or no inhibition is shown by aphidicolin (an inhibitor of eukaryotic DNA polymerase alpha), dideoxythymidine triphosphate (an inhibitor of eukaryotic DNA polymerases beta and gamma), nalidixic acid, or rifampicin. Ethidium bromide is a moderate inhibitor of DNA synthesis in the isolated chloroplast. Soluble extracts of chloroplasts will copy exogenously added recombinant plasmid circular DNA containing fragments of chloroplast DNA, and this reaction is strongly inhibited by ethidium bromide. Copying of the plasmid DNA takes place on the relaxed circular or linear forms of the DNA, but no specific initiation sites on the chloroplasts' DNA fragments of the recombinant plasmids have been detected. Our data are consistent with a repair mechanism operating in vitro but may also represent incomplete replicative DNA synthesis.     

Excision repair of DNA in nuclear extracts from the yeast Saccharomyces cerevisiae.

Excision repair of DNA is an important cellular response to DNA damage caused by a broad spectrum of physical and chemical agents. We have established a cell-free system in which damage-specific DNA repair synthesis can be demonstrated in vitro with nuclear extracts from the yeast Saccharomyces cerevisiae. Repair synthesis of UV-irradiated plasmid DNA was observed in a radiation dose-dependent manner and was unaffected by mutations in the RAD1, RAD2, RAD3, RAD4, RAD10, or APN1 genes. DNA damaged with cis-platin was not recognized as a substrate for repair synthesis. Further examination of the repair synthesis observed with UV-irradiated DNA revealed that it is dependent on the presence of endonuclease III-sensitive lesions in DNA, but not pyrimidine dimers. These observations suggest that the repair synthesis observed in yeast nuclear extracts reflects base excision repair of DNA. Our data indicate that the patch size of this repair synthesis is at least seven nucleotides. This system is expected to facilitate the identification of specific gene products which participate in base excision repair in yeast.     

Deoxyuridine triphosphate pools after polyoma virus infection

The synthesis of polyoma DNA in virus-infected 3T6 mouse fibroblasts is discontinuous with the intermediate formation of short Okazaki fragments. Hydroxyurea, an inhibitor of the enzyme ribonucleotide reductase, inhibits polyoma DNA synthesis, as measured by incorporation of radioactive thymidine. In the inhibited state, almost all incorporation occurs into short fragments. We investigated to what extent formation of short DNA fragments might be the result of incorporation of deoxyuridine triphosphate (dUTP) into DNA, followed by excision and repair reactions. We devised a sensitive enzymatic method for measuring dUTP in cell extracts which allows the determination of the dUTP pool when this pool amounts to between 0.1 and 2% of the dTTP pool. No dUTP was detected in growing mouse fibroblasts. After infection with polyoma virus cell extracts contained 0.4% dUTP (of dTTP) at the peak of DNA synthesis. Addition of hydroxyurea at this point led to a disappearance of dUTP. We conclude that dUTP incorporation can contribute only minimally to the generation of short fragments during polyoma DNA synthesis.     

Characterization of the DNA polymerase requirement of human base excision repair.

Base excision repair is one of the major mechanisms by which cells correct damaged DNA. We have developed an in vitro assay for base excision repair which is dependent on a uracil-containing DNA template. In this report, we demonstrate the fractionation of a human cell extract into two required components. One fraction was extensively purified and by several criteria shown to be identical to DNA polymerase beta (Polbeta). Purified, recombinant Polbeta efficiently substituted for this fraction. Escherichia coli PolI, mammalian Poldelta and to a lesser extent Polalpha and epsilon also functioned in this assay. We provide evidence that multiple polymerases function in base excision repair in human cell extracts. A neutralizing antibody to Polbeta, which inhibited repair synthesis catalyzed by pure Polbeta by approximately 90%, only suppressed repair in crude extracts by a maximum of approximately 70%. An inhibitor of Polbeta, ddCTP, decreased base excision repair in crude extracts by approximately 50%, whereas the Polalpha/delta/epsilon inhibitor, aphidicolin, reduced the reaction by approximately 20%. A combination of these chemical inhibitors almost completely abolished repair synthesis. These data suggest that Polbeta is the major base excision repair polymerase in human cells, but that other polymerases also contribute to a significant extent.     

Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts

Soluble extracts from human lymphoid cell lines that perform repair synthesis on covalently closed circular DNA containing pyrimidine dimers or psoralen adducts are described. Short patches of nucleotides are introduced by excision repair of damaged DNA in an ATP-dependent reaction. Extracts from xeroderma pigmentosum cell lines fail to act on damaged circular DNA, but are proficient in repair synthesis of ultraviolet-irradiated DNA containing incisions generated by Micrococcus luteus pyrimidine dimer-DNA glycosylase. Repair is defective in extracts from all xeroderma pigmentosum cell lines investigated, representing the genetic complementation groups A, B, C, D, H, and V. Mixing of cell extracts of group A and C origin leads to reconstitution of the DNA repair activity.     

Down-regulation of DNA repair synthesis at DNA single-strand interruptions in poly(ADP-ribose) polymerase-1 deficient murine cell extracts

The functional involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in the repair of DNA single- and double-strand breaks, DNA base damage, and related repair substrate intermediates remains unclear. Using an in vitro DNA repair assay and cell extracts derived from PARP-1 deficient or wild-type murine embryonic fibroblasts, we investigated the DNA synthesis and ligation steps associated with the rejoining of DNA single-strand interruptions containing 3'-OH, and either 5'-OH or 5'-P termini. Complete repair leading to DNA rejoining was similar between PARP-1 deficient cells and wild-type controls and poly(ADP-ribose) synthesis was, as expected, greatly reduced in PARP-1 deficient cell extracts. The incorporation of [32P]dCMP into repaired DNA at the site of a lesion was reduced two-three-fold in PARP-1 deficient cell extracts, demonstrating a decrease in repair patch size. Addition of purified PARP-1 to levels approximating those present in wild-type extracts did not stimulate DNA repair synthesis. We conclude that PARP-1 is not required for the efficient processing and rejoining of single-strand interruptions with defined 3'-OH and 5'-OH or 5'-P termini. Decreased DNA repair synthesis observed in PARP-1 deficient cell extracts is associated with reduced cellular expression of several factors required for long-patch base excision repair (BER), including FEN-1 and DNA ligase I.     

Aphidicolin-resistant and -sensitive base excision repair in wild-type and DNA polymerase beta-defective mouse cells

Several DNA polymerases (Pols) can add complementary bases at the gap created during the base excision repair (BER). To characterize the BER resynthesis step, the repair of a single abasic site by wild-type and Pol beta-defective mouse cell extracts was analysed in the presence of aphidicolin, a specific inhibitor of replicative Pols. We show that there is a competition between distributive and processive Pols for the nucleotide addition at the primer terminus. In wild-type cell extracts, the initial nucleotide insertion involves mainly Pol beta but the elongation step is carried out by a replicative Pol. Conversely, in Pol beta-null cell extracts the synthesis step is carried out by a replicative Pol without any switching to an auxiliary polymerase. We present evidence that short-patch repair synthesis occurs even in the absence of both Pol beta and replicative Pols. Exogeneously added purified human Pol lambda was unable to stimulate this back-up synthesis.     

Acetylation regulates DNA repair mechanisms in human cells

The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation.     

Efficient in vitro repair of 7-hydro-8-oxodeoxyguanosine by human cell extracts: involvement of multiple pathways.

To investigate the repair of oxidative damage in DNA, we have established an in vitro assay utilizing human lymphoblastoid whole cell extracts and plasmid DNA damaged by exposure to methylene blue and visible light. This treatment has been shown to produce predominantly 7-hydro-8-oxodeoxyguanosine (8-oxodG) in double-stranded DNA at low levels of modification. DNA containing 1. 6 lesions per plasmid is substrate for efficient repair synthesis by cell extracts. The incorporation of dGMP is 2.7 +/- 0.5 times greater than the incorporation of dCMP, indicating an average repair patch of 3-4 nucleotides. Damage-specific nicking occurs within 15 min, while resynthesis is slower. The incorporation of dGMP increases linearly, while the incorporation of dCMP exhibits a distinct lag. Extracts from xeroderma pigmentosum (XP) complementation groups A and B exhibit 25 and 40%, respectively, of the incorporation of dCMP compared with normal extracts, but extracts from an XP-D cell line exhibit twice the activity. These data suggest that the efficient repair of 8-oxodG lesions observed in human cell extracts involves more than one pathway of base excision repair.     

DNA mismatch repair detected in human cell extracts.

A system to study mismatch repair in vitro in HeLa cell extracts was developed. Preformed heteroduplex plasmid DNA containing two single base pair mismatches within the SupF gene of Escherichia coli was used as a substrate in a mismatch repair assay. Repair of one or both of the mismatches to the wild-type sequence was measured by transformation of a lac(Am) E. coli strain in which the presence of an active supF gene could be scored. The E. coli strain used was constructed to carry mutations in genes associated with mismatch repair and recombination (mutH, mutU, and recA) so that the processing of the heteroduplex DNA by the bacterium was minimal. Extract reactions were carried out by the incubation of the heteroduplex plasmid DNA in the HeLa cell extracts to which ATP, creatine phosphate, creatine kinase, deoxynucleotides, and a magnesium-containing buffer were added. Under these conditions about 1% of the mismatches were repaired. In the absence of added energy sources or deoxynucleotides, the activity in the extracts was significantly reduced. The addition of either aphidicolin or dideoxynucleotides reduced the mismatch repair activity, but only aphidicolin was effective in blocking DNA polymerization in the extracts. It is concluded that mismatch repair in these extracts is an energy-requiring process that is dependent on an adequate deoxynucleotide concentration. The results also indicate that the process is associated with some type of DNA polymerization, but the different effects of aphidicolin and dideoxynucleotides suggest that the mismatch repair activity in the extracts cannot simply be accounted for by random nick-translation activity alone.     

A role for the human single-stranded DNA binding protein HSSB/RPA in an early stage of nucleotide excision repair.

The human single-stranded DNA binding protein (HSSB/RPA) is involved in several processes that maintain the integrity of the genome including DNA replication, homologous recombination, and nucleotide excision repair of damaged DNA. We report studies that analyze the role of HSSB in DNA repair. Specific protein-protein interactions appear to be involved in the repair function of HSSB, since it cannot be replaced by heterologous single-stranded DNA binding proteins. Anti-HSSB antibodies that inhibit the ability of HSSB to stimulate DNA polymerase alpha also inhibit repair synthesis mediated by human cell-free extracts. However, antibodies that neutralize DNA polymerase alpha do not inhibit repair synthesis. Repair is sensitive to aphidicolin, suggesting that DNA polymerase epsilon or delta participates in nucleotide excision repair by cell extracts. HSSB has a role other than generally stimulating synthesis by DNA polymerases, as it does not enhance the residual damage-dependent background synthesis displayed by repair-deficient extracts from xeroderma pigmentosum cells. Significantly, when damaged DNA is incised by the Escherichia coli UvrABC repair enzyme, human cell extracts can carry out repair synthesis even when HSSB has been neutralized with antibodies. This suggests that HSSB functions in an early stage of repair, rather than exclusively in repair synthesis. A model for the role of HSSB in repair is presented.