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Results for “Excision Repair”

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PubMed2000

Expression of ERCC1 antisense RNA abrogates gemicitabine-mediated cytotoxic synergism with cisplatin in human colon tumor cells defective in mismatch repair but proficient in nucleotide excision repair.

Gemcitabine, or 2',2'-difluorodeoxycytidine (dFdC) is a new anticancer agent with significant activity against a broad spectrum of tumors either as a single agent or in combination with other active anticancer drugs. Studies in vitro and in vivo have demonstrated that dFdC produces cytotoxic synergism with cisplatin, or cis-diamminedi-choloroplatinum(II) (CDDP); however, the mechanism by which the synergism occurs has not been elucidated. We proposed that the nucleotide excision repair (NER) process, which is responsible for the cellular removal of CDDP-DNA adducts, may be a target for the mechanism of the cytotoxic synergism of dFdC and CDDP. Because the mismatch repair (MMR) pathway is involved in mediating CDDP cytotoxicity, making determination of the role of the NER in the cytotoxic synergism more complicated, and because tumors are often defective in MMR, we selected an NER-proficient, MMR-deficient, CP2.0 human colon carcinoma cell line as a model for this study. By an in vitro repair synthesis assay, we found that dFdC triphosphate (dFdCTP), the active metabolite of dFdC, inhibited the incorporation of [alpha-32P]dATP as well as the incorporation of [alpha-32P]dCTP, suggesting that the repair inhibition by dFdCTP does not result simply from competition for the incorporation site but rather is also due to prevention of chain elongation during the DNA resynthesis process. To determine whether the repair inhibition contributes to the cytotoxic synergism, we examined the effect of the constitutive expression of ERCC1 antisense RNA on the interaction of dFdC and CDDP. CP2.0 cells were transfected with pERCC1/AS, an ERCC1 antisense expression vector; eight hygromycine-resistant clones expressing various levels of the antisense RNA were selected for quantification of and correlation between the repair activity and cytotoxic synergism. The results show that stable expression of ERCC1 antisense RNA down-regulated the level of mRNA and repair activity; the down-regulation of the repair activity significantly correlated with the reduction of the cytotoxic synergism of the two agents. These data provide direct evidence to support the hypothesis that inhibition of the repair of CDDP-induced DNA lesions plays a critical role in dFdC-mediated cytotoxic synergism with CDDP in MMR-deficient tumor cells.

Antineoplastic Agents↗

PubMed1997

Mechanism of open complex and dual incision formation by human nucleotide excision repair factors.

During nucleotide excision repair in human cells, a damaged DNA strand is cleaved by two endonucleases, XPG on the 3' side of the lesion and ERCC1-XPF on the 5' side. These structure-specific enzymes act at junctions between duplex and single-stranded DNA. ATP-dependent formation of an open DNA structure of approximately 25 nt around the adduct precedes this dual incision. We investigated the mechanism of open complex formation and find that mutations in XPB or XPD, the DNA helicase subunits of the transcription and repair factor TFIIH, can completely prevent opening and dual incision in cell-free extracts. A deficiency in XPC protein also prevents opening. The absence of RPA, XPA or XPG activities leads to an intermediate level of strand separation. In contrast, XPF or ERCC1-defective extracts open normally and generate a 3' incision, but fail to form the 5' incision. This same repair defect was observed in extracts from human xeroderma pigmentosum cells with an alteration in the C-terminal domain of XPB, suggesting that XPB has an additional role in facilitating 5' incision by ERCC1-XPF nuclease. These data support a mechanism in which TFIIH-associated helicase activity and XPC protein catalyze initial formation of the key open intermediate, with full extension to the cleavage sites promoted by the other core nucleotide excision repair factors. Opening is followed by dual incision, with the 3' cleavage made first.

Adenosine Triphosphate↗

PubMed1994

Reconstitution of the DNA base excision-repair pathway.

BACKGROUND: The base excision-repair pathway is the major cellular defence mechanism against spontaneous DNA damage. The enzymes involved have been highly conserved during evolution. Base excision-repair has been reproduced previously with crude cell-free extracts of bacterial or human origin. To further our understanding of base excision-repair, we have attempted to reconstitute the pathway in vitro using purified enzymes. RESULTS: We report here the successful reconstitution of the base excision-repair pathway with five purified enzymes from Escherichia coli: uracil-DNA glycosylase, a representative of the DNA glycosylases that remove various lesions from DNA; the AP endonuclease IV that specifically cleaves at abasic sites; RecJ protein which excises a 5' terminal deoxyribose-phosphate residue; DNA polymerase I; and DNA ligase. The reaction proceeds with high efficiency in the absence of additional factors in the reconstituted system. Four of the enzymes are absolutely required for completion of the repair reaction. An unusual feature we have discovered is that the pathway branches after enzymatic incision at an abasic DNA site. RecJ protein is required for the major reaction, which involves replacement of only a single nucleotide at the damaged site; in its absence, an alternative pathway is observed, with generation of longer repair patches by the 5' nuclease function of DNA polymerase I. CONCLUSIONS: Repair of uracil in DNA is achieved by a very short-patch excision-repair process involving five different enzymes. No additional protein factors seem to be required. There is a minor, back-up pathway that uses replication factors to generate longer repair patches.

Bacterial Proteins↗

PubMed2000

Oxidative base damage to DNA: specificity of base excision repair enzymes.

Base excision repair (BER) is likely to be the main mechanism involved in the enzymatic restoration of oxidative base lesions within the DNA of both prokaryotic and eukaryotic cells. Emphasis was placed in early studies on the determination of the ability of several bacterial DNA N-glycosylases, including Escherichia coli endonuclease III (endo III) and formamidopyrimidine DNA N-glycosylase (Fpg), to recognize and excise several oxidized pyrimidine and purine bases. More recently, the availability of related DNA repair enzymes from yeast and human has provided new insights into the enzymatic removal of several.OH-mediated modified DNA bases. However, it should be noted that most of the earlier studies have involved globally modified DNA as the substrates. This explains, at least partly, why there is a paucity of accurate kinetic data on the excision rate of most of the modified bases. Interestingly, several oxidized pyrimidine and purine nucleosides have been recently inserted into defined sequence oligonucleotides. The use of the latter substrates, together with overexpressed DNA N-glycosylases, allows detailed studies on the efficiency of the enzymatic release of the modified bases. This was facilitated by the development of accurate chromatographic and mass spectrometric methods aimed at measuring oxidized bases and nucleosides. As one of the main conclusions, it appears that the specificity of both endo III and Fpg proteins is much broader than expected a few years ago.

DNA↗

PubMed1997

Rodent UV-sensitive mutant cell lines in complementation groups 6-10 have normal general excision repair activity.

Mammalian nucleotide excision repair is the primary enzymatic pathway for removing bulky lesions from DNA. The repair reaction involves three main steps: (i) dual incisions on both sides of the lesion; (ii) excision of the damaged base in an oligonucleotide 24-31 nt in length; (iii) filling in of the post-excision gap and ligation. We have developed assays that probe the individual steps of the reaction. Using these methods (assays for incision, excision and repair patch synthesis), we demonstrate that the mammalian excision nuclease system removes bulky lesions by incising mainly at the 22nd-25th phosphodiester bonds 5'and the 3rd-5th phosphodiester bonds 3'of the lesion, thus releasing oligonucleotides primarily 26-29 nt in length. The resulting excision gap is filled in by DNA polymerases delta and epsilon as revealed by the 'phosphorothioate repair patch assay'. When these assays were employed with cell-free extracts from the moderately UV-sensitive rodent mutants in complementation groups 6-10, we found that these mutants are essentially normal in all three steps of the repair reaction. This leads us to conclude that these cell lines have normal in vitro repair activities and that the defects in these mutants are most likely in genes controlling cellular functions not directly involved in general excision repair.

Animals↗

PubMed1995

DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract.

The G:U mismatch in genomic DNA mainly arises from deamination of cytosine residues and is repaired by the base excision repair pathway. We found that a bovine testis crude nuclear extract conducts uracil-initiated base excision repair in vitro. A 51-base pair synthetic DNA substrate containing a single G:U mismatch was used, and incorporation of dCMP during repair was exclusively to replace uracil. A neutralizing polyclonal antibody against DNA polymerase beta (beta-pol) inhibited the repair reaction. ddCTP also inhibited the repair reaction, whereas aphidicolin had no significant effect, suggesting that activity of beta-pol was required. Next, the base excision repair system was reconstituted using partially purified components. Several of the enzymatic activities required were resolved, such that DNA ligase and the uracil-DNA glycosylase/apurinic/apyrimidinic endonuclease activities were separated from the DNA polymerase requirement. We found that purified beta-pol could restore full DNA repair activity to the DNA polymerase-depleted fraction, whereas purified DNA polymerases alpha, delta, and epsilon could not. These results with purified proteins corroborated results obtained with the crude extract and indicate that beta-pol is responsible for the single-nucleotide gap filling reaction involved in this in vitro base excision repair system.

Animals↗

PubMed1984

Age-related changes in the excision repair capacity of Turbatrix aceti.

Excision repair capacity was measured in the free-living nematode Turbatrix aceti, a model aging system. Excision repair was assessed by both repair synthesis activity and the actual removal of pyrimidine dimers from the genome. The young organisms removed lesions more rapidly and completely than the old for all fluences tested. Repair synthesis began and peaked earlier for young nematodes than old. The data consistently indicated a decline in DNA excision repair capacity with age in the nematode.

Aging↗

PubMed2006

Is base excision repair a tumor suppressor mechanism?

The base excision repair pathway is critical for the removal of oxidized and methylated bases from the DNA. Much of this DNA damage arises endogenously, as a result of oxygen metabolism. Several proteins including DNA glycosylases, the APE1 endonuclease, DNA polymerase beta and DNA ligase, act in a highly regulated and coordinated manner during base excision repair to excise the base adducts from the DNA and restore the normal DNA sequence. Both germline and tumor-associated variants of genes encoding these proteins have been identified in humans. In many cases, the protein variant has been shown to have properties that could contribute to the development of cancer, suggesting that base excision repair acts as a tumor suppressor mechanism in humans. Limited epidemiological studies are consistent with this view. Our review of the literature indicates that additional laboratory and epidemiological studies of the role of base excision repair in cancer etiology is warranted.

Animals↗

PubMed1997

Bipartite substrate discrimination by human nucleotide excision repair.

Mammalian nucleotide excision repair (NER) eliminates carcinogen-DNA adducts by double endonucleolytic cleavage and subsequent release of 24-32 nucleotide-long single-stranded fragments. Here we manipulated the deoxyribose-phosphate backbone of DNA to analyze the mechanism by which damaged strands are discriminated as substrates for dual incision. We found that human NER is completely inactive on DNA duplexes containing single C4'-modified backbone residues. However, the same C4' backbone variants, which by themselves do not perturb complementary hydrogen bonds, induced strong NER reactions when incorporated into short segments of mispaired bases. No oligonucleotide excision was detected when DNA contained abnormal base pairs without concomitant changes in deoxyribose-phosphate composition. Thus, neither C4' backbone lesions nor improper base pairing stimulated human NER, but the combination of these two substrate alterations constituted an extremely potent signal for double DNA incision. In summary, we used C4'-modified backbone residues as molecular tools to dissect DNA damage recognition by human NER into separate components and identified a bipartite discrimination mechanism that requires changes in DNA chemistry with concurrent disruption of Watson-Crick base pairing.

Base Composition↗

PubMed1997

Mammalian DNA repair methyltransferases shield O4MeT from nucleotide excision repair.

O6-Methylguanine (O6MeG) and O4-methylthymine (O4MeT) are potentially mutagenic DNA lesions that cause G:C-->A:T and A:T-->G:C transition mutations by mispairing during DNA replication, and the repair of O6MeG and O4MeT by DNA repair methyltransferases (MTases) is therefore expected to prevent methylation-induced transitions. The efficiency of O6MeG and O4MeT repair by different MTases can vary by several hundred-fold and the aim of this study was to establish the biological consequences of such differences in the efficiency of repair. The ability of three microbial and two mammalian MTases to prevent methylation-induced G:C-->A:T and A:T-->G:C transitions is taken as a measure of their ability to repair O6MeG and O4MeT in vivo respectively. All five MTases give complete protection against G:C-->A:T transitions. However, while the microbial MTases give complete protection against A:T-->G:C transitions, the mammalian MTases actually sensitize cells to A:T-->G:C transitions. We hypothesize that the mammalian MTases bind O4MeT lesions in vivo but that, because they are extremely slow at subsequent methyl transfer, binding shields O4MeT from repair by the nucleotide excision repair pathway. Results are presented to support this hypothesis.

Animals↗

PubMed2003

The eukaryotic nucleotide excision repair pathway.

Nucleotide excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4 PPs). In this review, we describe the main protein players and the different sequential steps of the eukaryotic NER mechanism in human cells, from lesion recognition to damage removal and DNA synthesis. Studies on the dynamics of protein access to the damaged site, and the kinetics of lesion removal contribute to the knowledge of how the cells respond to genetic insult. DNA lesions as well as NER factors themselves are also implicated in changes in cell metabolism, influencing cell cycle progression or arrest, apoptosis and genetic instability. These changes are related to increased mutagenesis and carcinogenesis. Finally, the recent collection of genomic data allows one to recognize the high conservation and the evolution of eukaryotic NER. The distribution of NER orthologues in different organisms, from archaea to the metazoa, displays challenging observations. Some of NER proteins are widespread in nature, probably representing ancient DNA repair proteins, which are candidates to participate in a primitive NER mechanism.

Animals↗

PubMed2004

AP endonuclease and poly(ADP-ribose) polymerase-1 interact with the same base excision repair intermediate.

Base excision repair (BER) is a defense system that protects cells from deleterious effects secondary to modified or missing DNA bases. BER is known to involve apurinic/apyrimidinic endonuclease (APE) and DNA polymerase ss (ss-pol) among other enzymes, and recent studies have suggested that poly(ADP-ribose) polymerase-1 (PARP-1) also plays a role by virtue of its binding to BER intermediates. The main role of APE is cleavage of the DNA backbone at abasic sites, and the enzyme also can catalyze 3'- to 5'-exonuclease activity at the cleaved abasic site. Photocross-linking studies with mouse embryonic fibroblast (MEF) cell extracts described here indicated that APE and PARP-1 interact with the same APE-cleaved abasic site BER intermediate. The model BER intermediate used includes a synthetic abasic site sugar, i.e. tetrahydrofuran (THF), in place of the natural deoxyribose. APE cross-linked efficiently with this intermediate, but not with a molecule lacking the 5'-THF phosphate group, and the same property was demonstrated for PARP-1. The addition of purified APE to the MEF extract reduced the amount of PARP-1 cross-linked to the BER intermediate, suggesting that APE can compete with PARP-1. APE and PARP-1 were antagonists of each other in in vitro BER related reactions on this model BER intermediate. These results suggest that PARP-1 and APE can interact with the same BER intermediate and that competition between these two proteins may influence their respective BER related functions.

Animals↗

PubMed2004

Atypical protein kinase C stimulates nucleotide excision repair activity.

Nucleotide excision repair (NER) deals with bulky DNA damages. However, the regulation of this process is still unclear. Here, we show that both cell resistance to genotoxic agents that generate DNA lesions corrected by NER and in vitro NER activity are correlated with atypical protein kinase C (PKC) zeta expression levels. Moreover, repair intermediates are produced and eliminated more rapidly in UV-irradiated PKCzeta-overexpressing cells. The expression levels of XPC and hHR23B, two NER proteins, are correlated with PKCzeta expression. Altogether, these results strongly suggest that PKCzeta could act as a modulator of NER activity by regulating the expression of XPC/hHR23B heterodimer.

Base Sequence↗

PubMed2003

ATP-dependent selection between single nucleotide and long patch base excision repair.

DNA base excision repair (BER) constitutes a major mechanism to restore the integrity of the genome following modifications of nucleobases. Although it is well established that poly(ADP-ribosylation) facilitates BER, the mechanism of this stimulation has remained unknown. Previous observations suggested that poly(ADP-ribose), which is synthesised from NAD(+), could serve as a unique source of ATP required for the ligation step in BER. This pathway of ATP generation is thought to compensate ATP shortage and relies on the release of pyrophosphate during DNA repair synthesis. Here, we present evidence that, in situations of cellular energy depletion, the synthesis of poly(ADP-ribose) is indeed stimulated. Simultaneously, single nucleotide repair is reduced. Rather, the number of nucleotides incorporated by DNA polymerase beta (Pol beta) during DNA repair synthesis is increased. Using a reconstituted system including the recombinant BER proteins Pol beta, AP endonuclease 1 (APE 1), X-ray repair cross-complementing group-1 (XRCC1), DNA ligase III (Lig III), flap endonuclease 1 (FEN 1), and poly(ADP-ribose) polymerase-1 (PARP-1), it is demonstrated that in the absence of ATP, both long patch DNA synthesis by Pol beta and poly(ADP-ribosylation) catalysed by PARP-1 are stimulated. Consequently, the preferred use of either long patch or single nucleotide BER depends on the availability of ATP. It is proposed that long patch BER is required for ATP generation from poly(ADP-ribose) and, therefore, predominant under conditions of ATP shortage.

Adenosine Triphosphate↗

PubMed2004

Phosphorylation of XPB helicase regulates TFIIH nucleotide excision repair activity.

Nucleotide excision repair (NER) removes damage from DNA in a tightly regulated multiprotein process. The xeroderma pigmentosum group B (XPB) helicase subunit of TFIIH functions in NER and transcription. The serine 751 (S751) residue of XPB was found to be phosphorylated in vivo. This phosphorylation inhibits NER and the microinjection of a phosphomimicking XPB-S751E mutant is unable to correct the NER defect of XP-B cells. Conversely, XPB-S751 dephosphorylation or its substitution with alanine (S751A) restores NER both in vivo and in vitro. Surprisingly, phospho/dephosphorylation of S751 spares TFIIH-dependent transcription. Finally, the phosphorylation of XPB-S751 does not impair the TFIIH unwinding of the DNA around the lesion, but rather prevents the 5' incision triggered by the ERCC1-XPF endonuclease. These data support an additional role for XPB in promoting the incision of the damaged fragment and reveal a point of NER regulation on TFIIH without interference in its transcription activity.

Animals↗

PubMed2000

Spt4 modulates Rad26 requirement in transcription-coupled nucleotide excision repair.

The nucleotide excision repair machinery can be targeted preferentially to lesions in transcribed sequences. This mode of DNA repair is referred to as transcription-coupled repair (TCR). In yeast, the Rad26 protein, which is the counterpart of the human Cockayne syndrome B protein, is implicated specifically in TCR. In a yeast strain genetically deprived of global genome repair, a deletion of RAD26 renders cells UV sensitive and displays a defect in TCR. Using a genome-wide mutagenesis approach, we found that deletion of the SPT4 gene suppresses the rad26 defect. We show that suppression by the absence of Spt4 is specific for a rad26 defect and is caused by reactivation of TCR in a Rad26-independent manner. Spt4 is involved in the regulation of transcription elongation. The absence of this regulation leads to transcription that is intrinsically competent for TCR. Our findings suggest that Rad26 acts as an elongation factor rendering transcription TCR competent and that its requirement can be modulated by Spt4.

Antineoplastic Agents, Alkylating↗

PubMed2002

The C-terminal region of Escherichia coli UvrC contributes to the flexibility of the UvrABC nucleotide excision repair system.

Nucleotide excision repair in Escherichia coli involves formation of the UvrB-DNA complex and subsequent DNA incisions on either site of the damage by UvrC. In this paper, we studied the incision of substrates with different damages in varying sequence contexts. We show that there is not always a correlation between the incision efficiency and the stability of the UvrB-DNA complex. Both stable and unstable UvrB-DNA complexes can be efficiently incised. However some lesions that give rise to stable UvrB-DNA complexes do result in a very low incision. We present evidence that this poor incision is due to sterical hindrance of the damage itself. In its C-terminal region UvrC contains two helix-hairpin-helix (HhH) motifs. Mutational analysis shows that these motifs constitute one functional unit, probably folded as one structural unit; the (HhH)2 domain. This (HhH)2 domain was previously shown to be important for the 5' incision on a substrate containing a (cis-Pt).GG adduct, but not for 3' incision. Here we show that, mainly depending on the sequence context of the lesion, the (HhH)2 domain can be important for 3' and/or 5' incision. We propose that the (HhH)2 domain stabilises specific DNA structures required for the two incisions, thereby contributing to the flexibility of the UvrABC repair system.

Adenosine Triphosphatases↗

PubMed2005

Isolation of a small molecule inhibitor of DNA base excision repair.

The base excision repair (BER) pathway is essential for the removal of DNA bases damaged by alkylation or oxidation. A key step in BER is the processing of an apurinic/apyrimidinic (AP) site intermediate by an AP endonuclease. The major AP endonuclease in human cells (APE1, also termed HAP1 and Ref-1) accounts for >95% of the total AP endonuclease activity, and is essential for the protection of cells against the toxic effects of several classes of DNA damaging agents. Moreover, APE1 overexpression has been linked to radio- and chemo-resistance in human tumors. Using a newly developed high-throughput screen, several chemical inhibitors of APE1 have been isolated. Amongst these, CRT0044876 was identified as a potent and selective APE1 inhibitor. CRT0044876 inhibits the AP endonuclease, 3'-phosphodiesterase and 3'-phosphatase activities of APE1 at low micromolar concentrations, and is a specific inhibitor of the exonuclease III family of enzymes to which APE1 belongs. At non-cytotoxic concentrations, CRT0044876 potentiates the cytotoxicity of several DNA base-targeting compounds. This enhancement of cytotoxicity is associated with an accumulation of unrepaired AP sites. In silico modeling studies suggest that CRT0044876 binds to the active site of APE1. These studies provide both a novel reagent for probing APE1 function in human cells, and a rational basis for the development of APE1-targeting drugs for antitumor therapy.

Antineoplastic Agents↗