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PubMed2001

A multistep damage recognition mechanism for global genomic nucleotide excision repair.

A mammalian nucleotide excision repair (NER) factor, the XPC-HR23B complex, can specifically bind to certain DNA lesions and initiate the cell-free repair reaction. Here we describe a detailed analysis of its binding specificity using various DNA substrates, each containing a single defined lesion. A highly sensitive gel mobility shift assay revealed that XPC-HR23B specifically binds a small bubble structure with or without damaged bases, whereas dual incision takes place only when damage is present in the bubble. This is evidence that damage recognition for NER is accomplished through at least two steps; XPC-HR23B first binds to a site that has a DNA helix distortion, and then the presence of injured bases is verified prior to dual incision. Cyclobutane pyrimidine dimers (CPDs) were hardly recognized by XPC-HR23B, suggesting that additional factors may be required for CPD recognition. Although the presence of mismatched bases opposite a CPD potentiated XPC-HR23B binding, probably due to enhancement of the helix distortion, cell-free excision of such compound lesions was much more efficient than expected from the observed affinity for XPC-HR23B. This also suggests that additional factors and steps are required for the recognition of some types of lesions. A multistep mechanism of this sort may provide a molecular basis for ensuring the high level of damage discrimination that is required for global genomic NER.

Base Pair Mismatch↗
PubMed1998

Repair of DNA lesions induced by polycyclic aromatic hydrocarbons in human cell-free extracts: involvement of two excision repair mechanisms in vitro.

Polycyclic aromatic hydrocarbons (PAHs) are significant environmental pollutants representing an important risk factor in human cancers. DNA adducts formed by the ultimate carcinogens of PAHs are potentially toxic, mutagenic and carcinogenic. DNA repair represents an important defense system against these genotoxic insults. Using a human cell-free system we have examined repair of DNA lesions induced by several PAH dihydrodiol epoxides, including anti-(+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide, anti-(+/-)-benz[a]anthracene-trans-3,4-dihydrodiol-1,2-epoxide, anti-(+/-)-benz[a]anthracene-trans-8,9-dihydrodiol-10,11-epoxide, anti-(+/-)-benzo[b]fluoranthene-trans-9,10-dihydrodiol-11,12-epoxide and anti-(+/-)-chrysene-trans-1,2-dihydrodiol-3,4-epoxide. Effective repair of DNA damage induced by these five PAH metabolites was detected. Two distinct mechanisms of excision repair were observed. The major repair mechanism is nucleotide excision repair (NER). The other mechanism is independent of NER and correlated with the presence of apurinic/apyrimidinic sites in the damaged DNA, thus presumably reflecting base excision repair (BER). However, the contribution of BER to different PAH lesions varied in vitro. These results suggest the possibility that BER may also play an important role in repair of certain PAH-induced DNA lesions.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1999

Extended X-ray absorption fine structure evidence for a single metal binding domain in Xenopus laevis nucleotide excision repair protein XPA.

Nucleotide excision repair (NER) is an important cellular mechanism, conserved from bacteria to humans, responsible for eliminating multiple types of structurally distinct DNA lesions from the genome. The protein XPA appears to play a central role in NER, recognizing and/or verifying damaged DNA and recruiting other proteins, including RPA, ERCC1, and TFIIH, to repair the damage. Sequence analysis and genetic evidence suggest that zinc, which is essential for DNA binding, is associated with a C4-type motif, C-X2-C-X17-C-X2-C. Sequence analysis suggests that a second, H2C2-type zinc-binding motif may be present near the C-terminal. Seventy percent of the amino acid sequence of Xenopus laevis XPA (xXPA) is identical to human XPA and both putative zinc-binding motifs are conserved in all known XPA proteins. Electrospray ionization-mass spectroscopy data show that xXPA contains only one zinc atom per molecule. EXAFS spectra collected on full-length xXPA in frozen (77 K) 15% glycerol aqueous solution unequivocally show that the zinc atom is coordinated to four sulfur atoms with an average Zn--S bond length of 2.33 +/- 0.02 A. Together, the EXAFS and mass spectroscopy data indicate that xXPA contains just one C4-type zinc-binding motif.

Amino Acid Sequence↗
PubMed2006

TFIIH enzymatic activities in transcription and nucleotide excision repair.

Transcription and nucleotide excision repair (NER) are two major mechanisms in which the transcription factor TFIIH plays a crucial role. In order to investigate its function, we first described a fast and efficient purification protocol of TFIIH from either HeLa cells or patient cell lines, as well as various in vitro enzymatic assays set up in our laboratory. All these enzymatic assays have been adapted to work on immobilized DNA, a powerful tool allowing for sequential protein incubations in various buffer conditions, without destabilizing protein complexes bound to the DNA. Runoff transcription assays performed with either whole cell extract or highly purified factors underline the role of TFIIH helicases (XPB and XPD) in the RNA synthesis. Moreover, the requirement of XPB and XPD in NER can also be investigated with various assays corresponding to the different steps of this process. The DNA opening assay (permanganate footprint) highlights DNA unwinding of the double-stranded DNA fragment within the repair complex, whereas the dual incision assay allows for detection of the double cut on both sides of the lesion. The gap-filling reaction following the cuts can be monitored as well with a DNA resynthesis assay. Futhermore, the use of immobilized DNA is of great interest to study the detailed mechanism in which TFIIH plays a central role. This chapter describes the ATP-independent recruitment of TFIIH on the damaged DNA previously recognized by XPC-hHR23B and the sequential arrival and departure of the repair proteins within the NER complex.

Cell Line↗
PubMed2000

Damage recognition in nucleotide excision repair of DNA.

Nucleotide excision repair (NER) is found throughout nature, in eubacteria, eukaryotes and archaea. In human cells it is the main pathway for the removal of damage caused by UV light, but it also acts on a wide variety of other bulky helix-distorting lesions caused by chemical mutagens. An ongoing challenge is to understand how a site of DNA damage is located during NER and distinguished from non-damaged sites. This article reviews information on damage recognition in mammalian cells and the bacterium Escherichia coli. In mammalian cells the XPC-hHR23B, XPA, RPA and TFIIH factors may all have a role in damage recognition. XPC-hHR23B has the strongest affinity for damaged DNA in some assays, as does the similar budding yeast complex Rad4-Rad23. There is current discussion as to whether XPC or XPA acts first in the repair process to recognise damage or distortions. TFIIH may play a role in distinguishing the damaged strand from the non-damaged one, if translocation along a DNA strand by the TFIIH DNA helicases is interrupted by encountering a lesion. The recognition and incision steps of human NER use 15 to 18 polypeptides, whereas E. coli requires only three proteins to obtain a similar result. Despite this, many remarkable similarities in the NER mechanism have emerged between eukaryotes and bacteria. These include use of a distortion-recognition factor, a strand separating helicase to create an open preincision complex, participation of structure-specific endonucleases and the lack of a need for certain factors when a region containing damage is already sufficiently distorted.

Animals↗
PubMed2006

Distinct functions of the ubiquitin-proteasome pathway influence nucleotide excision repair.

The Rad23/Rad4 nucleotide excision repair (NER) protein complex functions at an early stage of the NER reaction, possibly promoting the recognition of damaged DNA. Here we show that Rad4 protein is ubiquitinated and degraded in response to ultraviolet (UV) radiation, and identify a novel cullin-based E3 ubiquitin ligase required for this process. We also show that this novel ubiquitin ligase is required for optimal NER. Our results demonstrate that optimal NER correlates with the ubiquitination of Rad4 following UV radiation, but not its subsequent degradation. Furthermore, we show that the ubiquitin-proteasome pathway (UPP) regulates NER via two distinct mechanisms. The first occurs independently of de novo protein synthesis, and requires Rad23 and a nonproteolytic function of the 19S regulatory complex of the 26S proteasome. The second requires de novo protein synthesis, and relies on the activity of the newly identified E3 ubiquitin ligase. These studies reveal that, following UV radiation, NER is mediated by nonproteolytic activities of the UPP, via the ubiquitin-like domain of Rad23 and UV radiation-induced ubiquitination of Rad4.

DNA Repair↗
PubMed1996

Double strand breaks in DNA inhibit nucleotide excision repair in vitro.

Nucleotide excision repair (NER) was measured in human cell extracts incubated with either supercoiled or linearized damaged plasmid DNA as repair substrate. NER, as quantified by the extent of repair synthesis activity, was reduced by up to 80% in the case of linearized plasmid DNA when compared with supercoiled DNA. An excess of undamaged linearized plasmid in the repair mixture did not interfere with DNA repair synthesis activity on a supercoiled damaged plasmid, indicating a cis-acting inhibiting effect. In contrast, gaps on circular or linearized plasmids were filled in identically by the DNA polymerases operating in the extracts. When the extent of damage-dependent incision activity was measured, a approximately 70% reduction of repair incision activity by human cell extract was observed on linearized damaged plasmids. Recessed, protruding, or blunt ends were similarly inhibitory. NER activity was partly restored when the extracts were preincubated with autoimmune human sera containing antibodies against the nuclear DNA end-binding heterodimer Ku. In addition, the inhibition of repair activity on linear damaged plasmids was released in extracts from rodent cells deficient in Ku activity but not in extracts from murine scid cells devoid of Ku-associated DNA-dependent kinase activity.

Animals↗
PubMed1999

Crystal structure of Thermus thermophilus HB8 UvrB protein, a key enzyme of nucleotide excision repair.

In the nucleotide excision repair system, UvrB plays a central role in damage recognition and DNA incision by interacting with UvrA and UvrC. We have determined the crystal structure of Thermus thermophilus HB8 UvrB at 1.9 A resolution. UvrB comprises four domains, two of which have an alpha/beta structure resembling the core domains of DNA and RNA helicases. Additionally, UvrB has an alpha-helical domain and a domain consisting of antiparallel beta-sheets (beta-domain). The sequence similarity suggests that the beta-domain interacts with UvrA. Based on the distribution of the conserved regions and the structure of the PcrA-DNA complex, a model for the UvrB-DNA complex is proposed.

Bacterial Proteins↗
PubMed2006

Complex formation with damage recognition protein Rad14 is essential for Saccharomyces cerevisiae Rad1-Rad10 nuclease to perform its function in nucleotide excision repair in vivo.

Nucleotide excision repair (NER) in eukaryotes requires the assembly of a large number of protein factors at the lesion site which then coordinate the dual incision of the damaged DNA strand. However, the manner by which the different protein factors are assembled at the lesion site has remained unclear. Previously, we have shown that in the yeast Saccharomyces cerevisiae, NER proteins exist as components of different protein subassemblies: the Rad1-Rad10 nuclease, for example, forms a tight complex with the damage recognition protein Rad14, and the complex of Rad1-Rad10-Rad14 can be purified intact from yeast cells. As the Rad1-Rad10 nuclease shows no specificity for binding UV lesions in DNA, association with Rad14 could provide an effective means for the targeting of Rad1-Rad10 nuclease to damage sites in vivo. To test the validity of this idea, here we identify two rad1 mutations that render yeast cells as UV sensitive as the rad1Delta mutation but which have no effect on the recombination function of Rad1. From our genetic and biochemical studies with these rad1 mutations, we conclude that the ability of Rad1-Rad10 nuclease to associate in a complex with Rad14 is paramount for the targeting of this nuclease to lesion sites in vivo. We discuss the implications of these observations for the means by which the different NER proteins are assembled at the lesion site.

DNA Damage↗
PubMed2000

Base excision repair is efficient in cells lacking poly(ADP-ribose) polymerase 1.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme that is activated by binding to DNA breaks induced by ionizing radiation or through repair of altered bases in DNA by base excision repair. Mice lacking PARP-1 and, in certain cases, the cells derived from these mice exhibit hypersensitivity to ionizing radiation and alkylating agents. In this study we investigated base excision repair in cells lacking PARP-1 in order to elucidate whether their augmented sensitivity to DNA damaging agents is due to an impairment of the base excision repair pathway. Extracts prepared from wild-type cells or cells lacking PARP-1 were similar in their ability to repair plasmid DNA damaged by either X-rays (single-strand DNA breaks) or by N:-methyl-N:'-nitro-N:-nitrosoguanidine (methylated bases). In addition, we demonstrated in vivo that PARP-1-deficient cells treated with N:-methyl-N:'-nitro-N:-nitrosoguanidine repaired their genomic DNA as efficiently as wild-type cells. Therefore, we conclude that cells lacking PARP-1 have a normal capacity to repair single-strand DNA breaks inflicted by X-irradiation or breaks formed during the repair of modified bases. We propose that the hypersensitivity of PARP-1 null mutant cells to gamma-irradiation and alkylating agents is not directly due to a defect in DNA repair itself, but rather results from greatly reduced poly(ADP-ribose) formation during base excision repair in these cells.

Animals↗
PubMed1987

Action of etoposide (VP-16-123) on human cells: no evidence for topoisomerase II involvement in excision repair of u.v.-induced DNA damage, nor for mitochondrial hypersensitivity in ataxia telangiectasia.

The excision repair of u.v. damage has been supposed to involve an initial action of DNA topoisomerase II, since some pre-incision step is sensitive to novobiocin, a topoisomerase II inhibitor. But novobiocin also affects mitochondrial structure and ATP metabolism, and this may account for its apparent inhibition of energy-dependent excision repair. We have investigated the effects of etoposide, another inhibitor of topoisomerase II, on u.v.-irradiated human cells: it is a more specific agent with no immediate side-effects on mitochondria. But etoposide is without effect on cellular excision repair, at the pre-incision stage or at the later stages of either DNA resynthesis or strand break ligation; nor does it potentiate cell killing after u.v. irradiation. The chromosome decondensation that accompanies incomplete excision repair in mitotic cells is likewise not greatly affected by etoposide. Therefore, if topoisomerase II is involved in excision repair or its regulation, it acts through a process that in whole cells is insensitive to etoposide. In ataxia telangiectasia cells, which are known to be hypersensitive to etoposide, the mitochondrial activities are not abnormally affected.

Ataxia Telangiectasia↗
PubMed1991

DNA excision repair in mammalian cell extracts.

The many genetic complementation groups of DNA excision-repair defective mammalian cells indicate the considerable complexity of the excision repair process. The cloning of several repair genes is taking the field a step closer to mechanistic studies of the actions and interactions of repair proteins. Early biochemical studies of mammalian DNA repair in vitro are now at hand. Repair synthesis in damaged DNA can be monitored by following the incorporation of radiolabelled nucleotides. Synthesis is carried out by mammalian cell extracts and is defective in extracts from cell lines derived from individuals with the excision-repair disorder xeroderma pigmentosum. Biochemical complementation of the defective extracts can be used to purify repair proteins. Repair of damage caused by agents including ultraviolet irradiation, psoralens, and platinating compounds has been observed. Neutralising antibodies against the human single-stranded DNA binding protein (HSSB) have demonstrated a requirement for this protein in DNA excision repair as well as in DNA replication.

Animals↗
PubMed1981

The relationship of DNA excision repair of ultraviolet-induced lesions to the maximum life span of mammals.

Physical and chemical agents present in the environment can potentially damage mammalian DNA. Such damage is known in some cases to be repaired by the process of DNA excision repair. This process has been extensively studied utilizing the repair of ultraviolet irradiation damage as a model system. In this study we have used this system and the 5-bromodeoxyuridine photolysis assay to measure DNA excision repair in cells derived from 21 mammalian species. We have attempted to relate the DNA repair proficiencies and the average size of the repaired regions seen in the cell cultures with the various maximum life spans of the mammals studied. There was an approximate linear correlation between life span of the mammals and the number of DNA excision repair sites measured 20-22 hours following ultraviolet irradiation of the cell cultures. Several deviations from the linear relationships were observed which remain largely unexplained. The size of the repaired regions was shown not to be related to the maximum life spans of the mammals tested.

Animals↗
PubMed2004

TFIIH contains a PH domain involved in DNA nucleotide excision repair.

The human general transcription factor TFIIH is involved in both transcription and DNA repair. We have identified a structural domain in the core subunit of TFIIH, p62, which is absolutely required for DNA repair activity through the nucleotide excision repair pathway. Using coimmunoprecipitation experiments, we showed that this activity involves the interaction between the N-terminal domain of p62 and the 3' endonuclease XPG, a major component of the nucleotide excision repair machinery. Furthermore, we reconstituted a functional TFIIH particle with a mutant of p62 lacking the N-terminal domain, showing that this domain is not required for assembly of the TFIIH complex and basal transcription. We solved its three-dimensional structure and found an unpredicted pleckstrin homology and phosphotyrosine binding (PH/PTB) domain, uncovering a new class of activity for this fold.

Amino Acid Sequence↗
PubMed1984

Induction by UV light of the SOS function sfiA in Escherichia coli strains deficient or proficient in excision repair.

The influence of the nucleotide excision repair system on the induction by UV irradiation of the SOS function sfiA has been investigated. The level of sfiA expression was monitored by means of a sfiA::lacZ operon fusion in both the wild-type strain and a uvrA mutant. We found that the initial steady rate of sfiA expression was proportional to the UV dose and was identical in uvr+ and uvrA backgrounds. This suggests that the initial steady rate of sfiA expression is determined by the initial number of lesions and before any effect of excision repair. We confirmed that after 2 h of expression the net synthesis of sfiA product is, for the same UV dose, about five times lower in uvr+ than in uvrA strains. We show that this is due to earlier repression of the SOS system in uvr+ than in uvrA strains and not to different initial rates.

DNA Repair↗
PubMed1993

Yeast excision repair gene RAD2 encodes a single-stranded DNA endonuclease.

In eukaryotes nucleotide excision repair of DNA damaged by ultraviolet radiation requires several gene products; defects in this process result in the cancer-prone syndrome xeroderma pigmentosum (XP) in humans. The RAD2 gene is one of at least seven genes indispensable for excision repair in the yeast Saccharomyces cerevisiae, and its encoded protein shares remarkable homology with the XP group-G gene product. Here we overproduce the RAD2-encoded protein in S. cerevisiae, purify it to near homogeneity, and show that RAD2 protein in the presence of magnesium degrades circular single-stranded DNA. The RAD2 endonuclease is specific for single-stranded DNA as it does not act on double-stranded DNA. Given the absolute requirement for RAD2 in the incision step of excision repair, our findings directly implicate RAD2 protein and its human homologue XPG protein as a catalytic component that incises the damaged DNA strand during excision repair. Furthermore, our results indicate that eukaryotes probably employ two distinct endonuclease activities to mediate the dual incision at the damage site.

DNA Repair↗
PubMed2006

Polymorphisms in estrogen bioactivation, detoxification and oxidative DNA base excision repair genes and prostate cancer risk.

To date, the potential impact of hormones on prostate cancer has predominantly focused on receptor-mediated events. However, catechol estrogens, if not inactivated by catechol-O-methyltransferase (COMT), can generate large quantities of reactive oxygen species (ROS). ROS may cause a spectrum of damage including oxidative DNA base lesions, which can lead to irreversible mutation(s) if they are not repaired by base excision repair (BER) systems. hOGG1 is a key enzyme in short patch BER because it recognizes and performs initial excision of the most common form of oxidative DNA base damage, 8-hydroxyguanine (8-oxo-dG). To investigate potential non-receptor-mediated estrogen effects, we evaluated the association between COMT Val158Met and hOGG1 Ser326Cys polymorphisms and prostate cancer in a family-based case-control study (439 prostate cancer cases, 479 brother controls). We observed no noteworthy associations between these polymorphisms and prostate cancer risk in the total study population. However, among men with more aggressive prostate cancer, the hOGG1 326 Cys/Cys genotype was inversely associated with disease (OR=0.30; 95% CI=0.09-0.98). Combining the lower activity CYP1B1 432 Leu/Leu or Leu/Val genotypes (which may decrease the level of catechol estrogens and ROS generated) with the hOGG1 326 Cys/Cys genotype and the XRCC1 399 Arg/Arg or Arg/Gln genotypes (which may enhance BER) resulted in an even further reduced risk in Caucasians with more aggressive disease (OR=0.09; 95% CI=0.01-0.56). Including the high-activity COMT 158Val allele to this combination also lowered aggressive prostate cancer risk but the effect was not as strong (OR=0.20; 95% CI=0.05-0.88). The decreased risk we observed with the hOGG1 326 Cys/Cys genotype confirms an earlier report and the further reduced risk found with the CYP1B1 (432 Leu/Leu or Leu/Val)-hOGG1 (326 Cys/Cys)-XRCC1 (Arg/Arg or Arg/Gln) genotype combination may lend new insights to the importance of ROS generated from non-receptor-mediated estrogenic mechanisms in more aggressive prostate cancer.

Aged↗