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Results for “nucleotide excision repair”

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At least 19 recordsLinked to original sources

PubMed2000

Contribution of base excision repair, nucleotide excision repair, and DNA recombination to alkylation resistance of the fission yeast Schizosaccharomyces pombe.

DNA damage is unavoidable, and organisms across the evolutionary spectrum possess DNA repair pathways that are critical for cell viability and genomic stability. To understand the role of base excision repair (BER) in protecting eukaryotic cells against alkylating agents, we generated Schizosaccharomyces pombe strains mutant for the mag1 3-methyladenine DNA glycosylase gene. We report that S. pombe mag1 mutants have only a slightly increased sensitivity to methylation damage, suggesting that Mag1-initiated BER plays a surprisingly minor role in alkylation resistance in this organism. We go on to show that other DNA repair pathways play a larger role than BER in alkylation resistance. Mutations in genes involved in nucleotide excision repair (rad13) and recombinational repair (rhp51) are much more alkylation sensitive than mag1 mutants. In addition, S. pombe mutant for the flap endonuclease rad2 gene, whose precise function in DNA repair is unclear, were also more alkylation sensitive than mag1 mutants. Further, mag1 and rad13 interact synergistically for alkylation resistance, and mag1 and rhp51 display a surprisingly complex genetic interaction. A model for the role of BER in the generation of alkylation-induced DNA strand breaks in S. pombe is discussed.

Adenine↗

PubMed1999

Overlapping specificities of base excision repair, nucleotide excision repair, recombination, and translesion synthesis pathways for DNA base damage in Saccharomyces cerevisiae.

The removal of oxidative damage from Saccharomyces cerevisiae DNA is thought to be conducted primarily through the base excision repair pathway. The Escherichia coli endonuclease III homologs Ntg1p and Ntg2p are S. cerevisiae N-glycosylase-associated apurinic/apyrimidinic (AP) lyases that recognize a wide variety of damaged pyrimidines (H. J. You, R. L. Swanson, and P. W. Doetsch, Biochemistry 37:6033-6040, 1998). The biological relevance of the N-glycosylase-associated AP lyase activity in the repair of abasic sites is not well understood, and the majority of AP sites in vivo are thought to be processed by Apn1p, the major AP endonuclease in yeast. We have found that yeast cells simultaneously lacking Ntg1p, Ntg2p, and Apn1p are hyperrecombinogenic (hyper-rec) and exhibit a mutator phenotype but are not sensitive to the oxidizing agents H2O2 and menadione. The additional disruption of the RAD52 gene in the ntg1 ntg2 apn1 triple mutant confers a high degree of sensitivity to these agents. The hyper-rec and mutator phenotypes of the ntg1 ntg2 apn1 triple mutant are further enhanced by the elimination of the nucleotide excision repair pathway. In addition, removal of either the lesion bypass (Rev3p-dependent) or recombination (Rad52p-dependent) pathway specifically enhances the hyper-rec or mutator phenotype, respectively. These data suggest that multiple pathways with overlapping specificities are involved in the removal of, or tolerance to, spontaneous DNA damage in S. cerevisiae. In addition, the fact that these responses to induced and spontaneous damage depend upon the simultaneous loss of Ntg1p, Ntg2p, and Apn1p suggests a physiological role for the AP lyase activity of Ntg1p and Ntg2p in vivo.

DNA Damage↗

PubMed2004

DNA repair in higher plants; photoreactivation is the major DNA repair pathway in non-proliferating cells while excision repair (nucleotide excision repair and base excision repair) is active in proliferating cells.

We investigated expression patterns of DNA repair genes such as the CPD photolyase, UV-DDB1, CSB, PCNA, RPA32 and FEN-1 genes by northern hybridization analysis and in situ hybridization using a higher plant, rice (Oryza sativa L. cv. Nipponbare). We found that all the genes tested were expressed in tissues rich in proliferating cells, but only CPD photolyase was expressed in non-proliferating tissue such as the mature leaves and elongation zone of root. The removal of DNA damage, cyclobutane pyrimidine dimers and (6-4) photoproducts, in both mature leaves and the root apical meristem (RAM) was observed after UV irradiation under light. In the dark, DNA damage in mature leaves was not repaired efficiently, but that in the RAM was removed rapidly. Using a rice 22K custom oligo DNA microarray, we compared global gene expression patterns in the shoot apical meristem (SAM) and mature leaves. Most of the excision repair genes were more strongly expressed in SAM. These results suggested that photoreactivation is the major DNA repair pathway for the major UV-induced damage in non-proliferating cells, while both photoreactivation and excision repair are active in proliferating cells.

Cell Division↗

PubMed2006

Single-nucleotide polymorphisms in base excision repair, nucleotide excision repair, and double strand break genes as markers for response to radiotherapy in patients with Stage I to II head-and-neck cancer.

PURPOSE: Polymorphisms in DNA repair genes can influence response to radiotherapy. We analyzed single-nucleotide polymorphisms (SNP) in nine DNA repair genes in 108 patients with head-and-neck cancer (HNSCC) who had received radiotherapy only. METHODS AND MATERIALS: From May 1993 to December 2004, patients with Stage I and II histopathologically confirmed HNSCC underwent radiotherapy. DNA was obtained from paraffin-embedded tissue, and SNP analysis was performed using a real-time polymerase chain reaction allelic discrimination TaqMan assay with minor modifications. RESULTS: Patients were 101 men (93.5%) and 7 (6.5%) women, with a median age of 64 years (range, 40 to 89 years). Of the patients, 76 (70.4%) patients were Stage I and 32 (29.6%) were Stage II. The XPF/ERCC1 SNP at codon 259 and XPG/ERCC5 at codon 46 emerged as significant predictors of progression (p = 0.00005 and 0.049, respectively) and survival (p = 0.0089 and 0.0066, respectively). Similarly, when variant alleles of XPF/ERCC1, XPG/ERCC5 and XPA were examined in combination, a greater number of variant alleles was associated with shorter time to progression (p = 0.0003) and survival (p = 0.0002). CONCLUSIONS: Genetic polymorphisms in XPF/ERCC1, XPG/ERCC5, and XPA may significantly influence response to radiotherapy; large studies are warranted to confirm their role in HNSCC.

Adult↗

PubMed1998

Physical interaction between components of DNA mismatch repair and nucleotide excision repair.

Nucleotide excision repair (NER) and DNA mismatch repair are required for some common processes although the biochemical basis for this requirement is unknown. Saccharomyces cerevisiae RAD14 was identified in a two-hybrid screen using MSH2 as "bait," and pairwise interactions between MSH2 and RAD1, RAD2, RAD3, RAD10, RAD14, and RAD25 subsequently were demonstrated by two-hybrid analysis. MSH2 coimmunoprecipitated specifically with epitope-tagged versions of RAD2, RAD10, RAD14, and RAD25. MSH2 and RAD10 were found to interact in msh3 msh6 and mlh1 pms1 double mutants, suggesting a direct interaction with MSH2. Mutations in MSH2 increased the UV sensitivity of NER-deficient yeast strains, and msh2 mutations were epistatic to the mutator phenotype observed in NER-deficient strains. These data suggest that MSH2 and possibly other components of DNA mismatch repair exist in a complex with NER proteins, providing a biochemical and genetical basis for these proteins to function in common processes.

Base Pair Mismatch↗

PubMed2002

Cho, a second endonuclease involved in Escherichia coli nucleotide excision repair.

Nucleotide excision repair removes damages from the DNA by incising the damaged strand on the 3' and 5' sides of the lesion. In Escherichia coli, the two incisions are made by the UvrC protein, which consists of two functional halves. The N-terminal half contains the catalytic site for 3' incision and the C-terminal half contains the residues involved in 5' incision. The genome of E. coli contains an SOS-inducible gene (ydjQ) encoding a protein that is homologous to the N-terminal half of UvrC. In this paper we show that this protein, which we refer to as Cho (UvrC homologue), can incise the DNA at the 3' side of a lesion during nucleotide excision repair. The incision site of Cho is located 4 nt further away from the damage compared with the 3' incision site of UvrC. Cho and UvrC bind to different domains of UvrB, which is probably the reason of the shift in incision position. Some damaged substrates that are poorly incised by UvrC are very efficiently incised by Cho. We propose that E. coli uses Cho for repair of such damages in vivo. Initially, most of the lesions in the cell will be repaired by the action of UvrC alone. Remaining damages, that for structural reasons obstruct the 3' incision by UvrC, will be repaired by the combined action of Cho (for 3' incision) and UvrC (for 5' incision).

Amino Acid Sequence↗

PubMed2004

Interactions between UvrA and UvrB: the role of UvrB's domain 2 in nucleotide excision repair.

Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism present in all kingdoms of life. UvrB is a central component of the bacterial NER system, participating in damage recognition, strand excision and repair synthesis. None of the three presently available crystal structures of UvrB has defined the structure of domain 2, which is critical for the interaction with UvrA. We have solved the crystal structure of the UvrB Y96A variant, which reveals a new fold for domain 2 and identifies highly conserved residues located on its surface. These residues are restricted to the face of UvrB important for DNA binding and may be critical for the interaction of UvrB with UvrA. We have mutated these residues to study their role in the incision reaction, formation of the pre-incision complex, destabilization of short duplex regions in DNA, binding to UvrA and ATP hydrolysis. Based on the structural and biochemical data, we conclude that domain 2 is required for a productive UvrA-UvrB interaction, which is a pre-requisite for all subsequent steps in nucleotide excision repair.

Adenosine Triphosphatases↗

PubMed1993

Nucleotide excision repair.

Nucleotide excision repair is the major DNA repair mechanism in all species tested. This repair system is the sole mechanism for removing bulky adducts from DNA, but it repairs essentially all DNA lesions, and thus, in addition to its main function, it plays a back-up role for other repair systems. In both pro- and eukaryotes nucleotide excision is accomplished by a multisubunit ATP-dependent nuclease. The excision nuclease of prokaryotes incises the eighth phosphodiester bond 5' and the fourth or fifth phosphodiester bond 3' to the modified nucleotide and thus excises a 12-13-mer. The excision nuclease of eukaryotes incises the 22nd, 23rd, or 24th phosphodiester bond 5' and the fifth phosphodiester bond 3' to the lesion and thus removes the adduct in a 27-29-mer. A transcription repair coupling factor encoded by the mfd gene in Escherichia coli and the ERCC6 gene in humans directs the excision nuclease to RNA polymerase stalled at a lesion in the transcribed strand and thus ensures preferential repair of this strand compared to the nontranscribed strand.

Animals↗

PubMed2001

A compromised yeast RNA polymerase II enhances UV sensitivity in the absence of global genome nucleotide excision repair.

Nucleotide excision repair is the major pathway responsible for removing UV-induced DNA damage, and is therefore essential for cell survival following exposure to UV radiation. In this report, we have assessed the contributions of some components of the RNA polymerase II (Pol II) transcription machinery to UV resistance in Saccharomyces cerevisiae. Deletion of the gene encoding the Pol II elongation factor TFIIS (SII) resulted in enhanced UV sensitivity, but only in the absence of global genome repair dependent on the RAD7 and RAD16 genes, a result seen previously with deletions of RAD26 and RAD28, yeast homologs of the human Cockayne syndrome genes CSB and CSA, respectively. A RAD7/16-dependent reduction in survival after UV irradiation was also seen in the presence of mutations in RNA Pol II that confer a defect in its response to SII, as well as with other mutations which reside in regions of the largest subunit of Pol II not involved in SII interactions. Indeed, an increase in UV sensitivity was achieved by simply decreasing the steadystate level of RNA Pol II. Truncation of the C-terminal domain and other RNA Pol II mutations conferred sensitivity to the ribonucleotide reductase inhibitor hydroxyurea and induction of RNR1 and RNR2 mRNAs after UV irradiation was attenuated in these mutant cells. That UV sensitivity can be a consequence of mutations in the RNA Pol II machinery in yeast cells suggests that alterations in transcriptional programs could underlie some of the pathophysiological defects seen in the human disease Cockayne syndrome.

Adenosine Triphosphatases↗

PubMed2004

The yeast Rad7/Rad16/Abf1 complex generates superhelical torsion in DNA that is required for nucleotide excision repair.

Nucleotide excision repair (NER) in eukaryotes removes DNA base damage as an oligonucleotide in a complex series of reactions. The nature of the dual incision reactions on either side of the damaged base has been extensively investigated. However, the precise mechanism of cleavage of the phosphodiester backbone of the DNA by the NER endonucleases and how this relates to removal of the damage-containing oligonucleotide during the excision process has not been determined. We previously isolated a stable heterotrimeric complex of Rad7/Rad16/Abf1 from yeast which functions in the conserved global genome repair (GGR) pathway. GGR removes lesions from DNA that is not actively transcribing. We have shown previously that the Rad7/Rad16/Abf1 heterotrimer is required to observe DNA repair synthesis and oligonucleotide excision during in vitro NER, but not needed to detect NER-dependent incision in such reactions. Here we report that this protein complex generates superhelicity in DNA through the catalytic activity of the Rad16 component. The torsion generated in the DNA by this complex is necessary to remove the damage-containing oligonucleotide during NER--a process referred to as excision. We conclude that in yeast the molecular mechanism of NER includes the generation of superhelical torsion in DNA.

Adenosine Triphosphatases↗

PubMed2000

Genomic heterogeneity of nucleotide excision repair.

Nucleotide excision repair (NER) is one of the major cellular pathways that removes bulky DNA adducts and helix-distorting lesions. The biological consequences of defective NER in humans include UV-light-induced skin carcinogenesis and extensive neurodegeneration. Understanding the mechanism of the NER process is of great importance as the number of individuals diagnosed with skin cancer has increased considerably in recent years, particularly in the United States. Rapid progress made in the DNA repair field since the early 1980s has revealed the complexity of NER, which operates differently in different genomic regions. The genomic heterogeneity of repair seems to be governed by the functional compartmentalization of chromatin into transcriptionally active and inactive domains in the nucleus. Two sub-pathways of NER remove UV-induced photolesions: (I) Global Genome Repair (GGR) and (II) Transcription Coupled Repair (TCR). GGR is a random process that occurs slowly, while the TCR, which is tightly linked to RNA polymerase II transcription, is highly specific and efficient. The efficiency of these pathways is important in avoiding cancer and genomic instability. Studies with cell lines derived from Cockayne syndrome (CS) and Xeroderma pigmentosum (XP) group C patients, that are defective in the NER sub-pathways, have yielded valuable information regarding the genomic heterogeneity of DNA repair. This review deals with the complexity of repair heterogeneity, its mechanism and interacting molecular pathways as well as its relevance in the maintenance of genomic integrity.

Animals↗

PubMedDate not supplied

Role of ATP hydrolysis by UvrA and UvrB during nucleotide excision repair.

Nucleotide excision repair in eubacteria is a process that repairs DNA damages by the removal of a 12-13-mer oligonucleotide containing the lesion. Recognition and cleavage of the damaged DNA is a multistep ATP-dependent reaction that requires the UvrA, UvrB and UvrC proteins. Both UvrA and UvrB are ATPases, with UvrA having two ATP binding sites which have the characteristic signature of the family of ABC proteins and UvrB having one ATP binding site that is structurally related to that of helicases.

Adenosine Triphosphatases↗

PubMed2005

Structural insights into the first incision reaction during nucleotide excision repair.

Nucleotide excision repair is a highly conserved DNA repair mechanism present in all kingdoms of life. The incision reaction is a critical step for damage removal and is accomplished by the UvrC protein in eubacteria. No structural information is so far available for the 3' incision reaction. Here we report the crystal structure of the N-terminal catalytic domain of UvrC at 1.5 A resolution, which catalyzes the 3' incision reaction and shares homology with the catalytic domain of the GIY-YIG family of intron-encoded homing endonucleases. The structure reveals a patch of highly conserved residues surrounding a catalytic magnesium-water cluster, suggesting that the metal binding site is an essential feature of UvrC and all GIY-YIG endonuclease domains. Structural and biochemical data strongly suggest that the N-terminal endonuclease domain of UvrC utilizes a novel one-metal mechanism to cleave the phosphodiester bond.

Amino Acid Sequence↗

PubMed2001

Centrosome protein centrin 2/caltractin 1 is part of the xeroderma pigmentosum group C complex that initiates global genome nucleotide excision repair.

Nucleotide excision repair (NER) is carried out by xeroderma pigmentosum (XP) factors. Before the excision reaction, DNA damage is recognized by a complex originally thought to contain the XP group C responsible gene product (XPC) and the human homologue of Rad23 B (HR23B). Here, we show that centrin 2/caltractin 1 (CEN2) is also a component of the XPC repair complex. We demonstrate that nearly all XPC complexes contain CEN2, that CEN2 interacts directly with XPC, and that CEN2, in cooperation with HR23B, stabilizes XPC, which stimulates XPC NER activity in vitro. CEN2 has been shown to play an important role in centrosome duplication. Thus, those findings suggest that the XPC-CEN2 interaction may reflect coupling of cell division and NER.

Calcium-Binding Proteins↗

PubMed1999

Crystal structure of UvrB, a DNA helicase adapted for nucleotide excision repair.

Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism. NER systems recognize the damaged DNA strand, cleave it on both sides of the lesion, remove and newly synthesize the fragment. UvrB is a central component of the bacterial NER system participating in damage recognition, strand excision and repair synthesis. We have solved the crystal structure of UvrB in the apo and the ATP-bound forms. UvrB contains two domains related in structure to helicases, and two additional domains unique to repair proteins. The structure contains all elements of an intact helicase, and is evidence that UvrB utilizes ATP hydrolysis to move along the DNA to probe for damage. The location of conserved residues and structural comparisons allow us to predict the path of the DNA and suggest that the tight pre-incision complex of UvrB and the damaged DNA is formed by insertion of a flexible beta-hairpin between the two DNA strands.

Adenosine Triphosphate↗

PubMed2004

Roles of Rad23 protein in yeast nucleotide excision repair.

Nucleotide excision repair (NER) removes many different types of DNA lesions. Most NER proteins are indispensable for repair. In contrast, the yeast Rad23 represents a class of accessory NER proteins, without which NER activity is reduced but not eliminated. In mammals, the complex of HR23B (Rad23 homolog) and XPC (yeast Rad4 homolog) has been suggested to function in the damage recognition step of NER. However, the precise function of Rad23 or HR23B in NER remains unknown. Recently, it was suggested that the primary function of RAD23 protein in NER is its stabilization of XPC protein. Here, we tested the significance of Rad23-mediated Rad4 stabilization in NER, and analyzed the repair and biochemical activities of purified yeast Rad23 protein. Cellular Rad4 was indeed stabilized by Rad23 in the absence of DNA damage. Persistent overexpression of Rad4 in rad23 mutant cells, however, largely failed to complement the ultraviolet sensitivity of the mutant. Consistently, deficient NER in rad23 mutant cell extracts could not be complemented by purified Rad4 protein in vitro. In contrast, partial complementation was observed with purified Rad23 protein. Specific complementation to the level of wild-type repair was achieved by adding purified Rad23 together with small amounts of Rad4 protein to rad23 mutant cell extracts. Purified Rad23 protein was unable to bind to DNA, but stimulated the binding activity of purified Rad4 protein to N-acetyl-2-aminofluorene-damaged DNA. These results support two roles of Rad23 protein in NER: (i) its direct participation in the repair biochemistry, possibly due to its stimulatory activity on Rad4-mediated damage binding/recognition; and (ii) its stabilization of cellular Rad4 protein.

DNA Repair↗

PubMed1999

Yeast autonomously replicating sequence binding factor is involved in nucleotide excision repair.

Nucleotide excision repair (NER) in yeast is effected by the concerted action of a large complex of proteins. Recently, we identified a stable subcomplex containing the yeast Rad7 and Rad16 proteins. Here, we report the identification of autonomously replicating sequence binding factor 1 (ABF1) as a component of the Rad7/Rad16 NER subcomplex. Yeast ABF1 protein is encoded by an essential gene required for DNA replication, transcriptional regulation, and gene silencing. We show that ABF1 plays a direct role in NER in vitro. Additionally, consistent with a role of ABF1 protein in NER in vivo, we show that certain temperature-sensitive abf1 mutant strains that are defective in DNA replication are specifically defective in the removal of photoproducts by NER and are sensitive to killing by ultraviolet (UV) radiation. These studies define a novel and unexpected role for ABF1 protein during NER in yeast.

Adenosine Triphosphatases↗

PubMed2004

The NEF4 complex regulates Rad4 levels and utilizes Snf2/Swi2-related ATPase activity for nucleotide excision repair.

Nucleotide excision repair factor 4 (NEF4) is required for repair of nontranscribed DNA in Saccharomyces cerevisiae. Rad7 and the Snf2/Swi2-related ATPase Rad16 are NEF4 subunits. We report previously unrecognized similarity between Rad7 and F-box proteins. Rad16 contains a RING domain embedded within its ATPase domain, and the presence of these motifs in NEF4 suggested that NEF4 functions as both an ATPase and an E3 ubiquitin ligase. Mutational analysis provides strong support for this model. The Rad16 ATPase is important for NEF4 function in vivo, and genetic analysis uncovered new interactions between NEF4 and Rad23, a repair factor that links repair to proteasome function. Elc1 is the yeast homologue of a mammalian E3 subunit, and it is a novel component of NEF4. Moreover, the E2s Ubc9 and Ubc13 were linked to the NEF4 repair pathway by genetic criteria. Mutations in NEF4 or Ubc13 result in elevated levels of the DNA damage recognition protein Rad4 and an increase in ubiquitylated species of Rad23. As Rad23 also controls Rad4 levels, these results suggest a complex system for globally regulating repair activity in vivo by controlling turnover of Rad4.

Adenosine Triphosphatases↗