Nucleotide excision repair (NER) functions to remove DNA damage caused by ultraviolet light and by other agents that distort the DNA helix. The NER machinery has been conserved in structure and function from yeast to humans, and in humans, defective NER is the underlying cause of the cancer-prone disease xeroderma pigmentosum. Here, we reconstitute the incision reaction of NER in Saccharomyces cerevisiae using purified protein factors. The Rad14 protein, the Rad4-Rad23 complex, the Rad2 nuclease, the Rad1-Rad10 nuclease, replication protein A, and the RNA polymerase II transcription factor TFIIH were purified to near homogeneity from yeast. We show that these protein factors are both necessary and sufficient for dual incision of DNA damaged by either ultraviolet light or N-acetoxy-2-aminoacetylfluorene. Incision in the reconstituted system requires ATP, which cannot be substituted by adenosine 5'-O-(3-thiotriphosphate), suggesting that the hydrolysis of ATP is indispensable for the incision reaction. The excision DNA fragments formed as a result of dual incision are in the 24-27-nucleotide range.

DNA mismatch repair plays a key role in the maintenance of genetic fidelity. Mutations in the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. The proliferating cell nuclear antigen (PCNA) is essential for DNA replication, where it acts as a processivity factor. Here, we identify a point mutation, pol30 -104, in the Saccharomyces cerevisiae POL30 gene encoding PCNA that increases the rate of instability of simple repetitive DNA sequences and raises the rate of spontaneous forward mutation. Epistasis analyses with mutations in mismatch repair genes MSH2, MLH1, and PMS1 suggest that the pol30 -104 mutation impairs MSH2/ MLH1/PMS1-dependent mismatch repair, consistent with the hypothesis that PCNA functions in mismatch repair. MSH2 functions in mismatch repair with either MSH3 or MSH6, and the MSH2-MSH3 and MSH2-MSH6 heterodimers have a role in the recognition of DNA mismatches. Consistent with the genetic data, we find specific interaction of PCNA with the MSH2-MSH3 heterodimer.In both prokaryotes and eukaryotes, defects in DNA mismatch repair cause elevated spontaneous mutation rates and increased instability of simple repeat DNA sequences. Mutations in any of the human mismatch repair genes hMSH2, hMLH1, hPMS1, and hPMS2 are associated with hereditary nonpolyposis colorectal cancer. Cell lines from these cancers are defective in DNA mismatch repair and display increased levels of spontaneous mutations and frequent alterations of microsatellite repeat sequences (1, 2).Epistasis analyses in yeast have suggested that MSH2 protein functions in conjunction with MSH3 or MSH6 protein in mismatch recognition. Genetic and biochemical studies in both yeast and humans have further indicated that the MSH2-MSH3 and MSH2-MSH6 complexes differ in substrate specificities. In yeast, mutations in MSH3 cause an increase in instability of microsatellite tracts but have little effect on single-base mispairs, whereas mutations in MSH6 have a more prominent effect on the incidence of single-base mispairs than on microsatellite tract instability (3-5). From these and other genetic observations, it has been inferred that MSH2-MSH3 complex is more proficient in the removal of insertion-deletion mismatches of two or more nucleotides (4), whereas MSH2-MSH6 is better at removing single nucleotide mismatches (4, 5). Human cell lines defective in the MSH6 component of the MSH2-MSH6 heterodimer hMutS␣ exhibit a selective loss in the repair of base-base and single-nucleotide insertion-deletion mismatches; the repair of two-, three-, and four-nucleotide insertion-deletion mismatches is reduced 2-4-fold in these cell lines (6, 7). Consistent with genetic observations, hMutS␣ binds a G/T mismatch or a one nucleotide insertion-deletion mismatch with high efficiency (6). By contrast, the yeast MSH2-MSH3 heterodimer exhibits little affinity for a G/T mismatch but binds insertion-deletion mismatches with high specificity (8). The manner by which PMS1 and MLH1 function in mismatch...

The RAD25 gene of Saccharomyces cerevisiae functions in nucleotide excision repair of ultraviolet-damaged DNA and is also required for cell viability. The RAD25 protein shows remarkable homology to the protein encoded by the human nucleotide-excision-repair gene XPB (ERCC3), mutations in which cause the cancer-prone disease xeroderma pigmentosum and also Cockayne's syndrome. Here we purify RAD25 protein from S. cerevisiae and show that it contains single-stranded DNA-dependent ATPase and DNA helicase activities. Extract from the conditional lethal mutant rad25-ts24 exhibits a thermolabile transcriptional defect which can be corrected by the addition of RAD25 protein, indicating a direct and essential role of RAD25 in RNA polymerase II transcription. The protein encoded by the rad25799am allele is defective in DNA repair but is proficient in RNA polymerase II transcription, indicating that RAD25 DNA-repair activity is separable from its transcription function. The rad25 Arg-392 encoded product, which contains a mutation in the ATP-binding motif, is defective in RNA polymerase II transcription, suggesting that the RAD25-encoded DNA helicase functions in DNA duplex opening during transcription initiation.

Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the involvement of a large number of protein factors in nucleotide excision repair (NER) of UV-damaged DNA. However, how MMS19 affects this process has remained unclear. Here, we report on the isolation of the MMS19 gene and the determination of its role in NER and other cellular processes. Genetic and biochemical evidence indicates that besides its function in NER, MMS19 also affects RNA polymerase II (Pol II) transcription. mms19delta cells do not grow at 37 degrees C, and mutant extract exhibits a thermolabile defect in Pol II transcription. Thus, Mms19 protein resembles TFIIH in that it is required for both transcription and DNA repair. However, addition of purified Mms19 protein does not alleviate the transcriptional defect of the mms19delta extract, nor does it stimulate the incision of UV-damaged DNA reconstituted from purified proteins. Interestingly, addition of purified TFIIH corrects the transcriptional defect of the mms19delta extract. Mms19 is, however, not a component of TFIIH or of Pol II holoenzyme. These and other results suggest that Mms19 affects NER and transcription by influencing the activity of TFIIH as an upstream regulatory element. It is proposed that mutations in the human MMS19 counterpart could result in syndromes in which both NER and transcription are affected.

The RAD3 gene of Saccharomyces cerevisiae is required for excision repair of ultraviolet-damaged DNA and is essential for cell viability. The RAD3-encoded protein shares a high degree of homology with the human ERCC2(XPD) gene product. Mutations in XPD, besides causing the cancer-prone syndrome xeroderma pigmentosum, can also result in Cockayne's syndrome and trichothiodystrophy. To investigate the role of RAD3 in viability, we examined here the effect of a recessive, temperature-sensitive (ts) conditional lethal mutation of the gene on transcription by RNA polymerase II. Upon transfer to the restrictive temperature, the rad3-ts mutant rapidly ceases growth and poly(A)+ RNA synthesis is inhibited drastically. Messenger RNA levels of all the genes examined, HIS3, TRP3, STE2, MET19, RAD23, CDC7, CDC9 and ACT1, decline rapidly upon loss of RAD3 activity. The synthesis of heat-shock-inducible HSP26 mRNA and galactose-inducible GAL7 and GAL10 mRNAs is also drastically inhibited in the rad3-ts mutant at the restrictive temperature. The RNA polymerase II transcriptional activity in extract from the rad3-ts14 strain is thermolabile, and this in vitro transcriptional defect can be fully corrected by the addition of homogeneous RAD3 protein. These findings indicate that RAD3 protein has a direct and essential role in RNA polymerase II transcription.

In eukaryotes, nucleotide excision repair of ultraviolet light-damaged DNA is a highly intricate process that requires a large number of evolutionarily conserved protein factors. Genetic studies in the yeast Saccharomyces cerevisiae have indicated a specific role of the RAD7 and RAD16 genes in the repair of transcriptionally inactive DNA. Here we show that the RAD7-and RAD16-encoded products exist as a complex of 1:1 stoichiometry, exhibiting an apparent dissociation constant (K d ) of <4 ؋ 10 ؊10 M. The Rad7-Rad16 complex has been purified to near homogeneity in this study and is shown to bind, in an ATP-dependent manner and with high specificity, to DNA damaged by ultraviolet light. Importantly, inclusion of the Rad7-Rad16 complex in the in vitro nucleotide excision repair system that consists entirely of purified components results in a marked stimulation of damage specific incision. Thus, Rad7-Rad16 complex is the ATP-dependent DNA damage sensor that specifically functions with the ensemble of nucleotide excision repair factor (NEF) 1, NEF2, NEF3, and replication protein A in the repair of transcriptionally inactive DNA. We name this novel complex of Rad7 and Rad16 proteins NEF4.In eukaryotes, nucleotide excision repair (NER) 1 of ultraviolet light-damaged DNA occurs by dual incision of the DNA strand that contains the UV lesion, excising the damage in the form of a short DNA fragment ϳ30 nucleotides in length (1). Mutational inactivation of NER in humans results in the cancer-prone syndrome xeroderma pigmentosum, which underscores the importance of this repair system in neutralizing the genotoxicity of UV light. The dual incision event in NER is accomplished by the concerted action of a large number of evolutionarily conserved proteins, and our studies in the yeast Saccharomyces cerevisiae have indicated an organization of these proteins into distinct subassemblies: nucleotide excision repair factor I, or NEF1, consisting of the damage recognition protein Rad14 and the Rad1-Rad10 endonuclease (2), NEF2, containing the Rad4 and Rad23 proteins (3), and NEF3, comprising the endonuclease Rad2 together with the RNA polymerase II transcription factor TFIIH (4). The combination of NEF1, NEF2, NEF3, and the heterotrimeric replication protein A (RPA) is sufficient for ATP-dependent dual incision to occur, indicating that the basic NER machinery is composed of these protein subassemblies (3). Dual incision of UV-damaged DNA can also be accomplished by combining the human equivalents of the aforementioned yeast repair proteins (5-7).At the genomic level, two functionally distinct modes of NER have been described, the first concerns with the repair of the transcribed strand in transcriptionally active chromosomal DNA, which involves the stalling of RNA polymerase II at the DNA lesion, and requires the CSA and CSB gene products (8). The second mode of excision repair is specific for the nontranscribed strand and for genomic regions that are transcriptionally inactive. Interestingly, in the rad7 and rad16 mutants of S. c...

Replication protein A (RPA), a heterotrimeric protein of 70-, 32-, and 14-kDa subunits, is an essential factor for DNA replication. Biochemical studies with human and yeast RPA have indicated that it is a DNA-binding protein that has higher affinity for single-stranded DNA. Interestingly, in vitro nucleotide excision repair studies with purified protein components have shown an absolute requirement for RPA in the incision of UV-damaged DNA. Here we use a mobility shift assay to demonstrate that human RPA binds a UV damaged duplex DNA fragment preferentially. Complex formation between RPA and the UV-irradiated DNA is not affected by prior enzymatic photo-reactivation of the DNA, suggesting an affinity of RPA for the (6-4) photoproduct. We also show that Mg 2؉ in the millimolar range is required for preferential binding of RPA to damaged DNA. These findings identify a novel property of RPA and implicate RPA in damage recognition during the incision of UV-damaged DNA.

Xeroderma pi um (XP) ATPase/lONA helicase activity in viability.In human, xeroderma pigmentosum (XP) is an autosomal recessive disorder, and cells from XP individuals are defective in the incision step of excision repair. Cell fusion studies have indicated the existence of 7 excision-deficient XP complementation groups, A-G (1-3); in addition, at least 10 complementation groups have been identified among UVsensitive excision-defective rodent cell lines (4-6). Five human genes-ERCCI, ERCC2, ERCC3, ERCC5, and ERCC6-have been cloned by cross-complementation of UV sensitivity of rodent cell lines (7-11). In addition, the human XPAC gene was isolated by complementation of the excision defect of XP-A cell lines (12). ERCCI does not complement any of the XP cell lines (13), whereas ERCC2 and ERCC3 restore normal excision repair to XP-D and XP-B mutant cell lines (9,14), respectively, indicating some degree of overlap between the XP and ERCC genes.At least 10 genes are involved in excision repair in the yeast Saccharomyces cerevisiae. Mutations in 6 of these genes-RADI, RAD2, RAD3, RAD4, RADIO, and RAD14-cause extreme UV sensitivity and a defect in the incision step of excision repair (15,16). The other four genes-RAD7, RAD16, RAD23, and MMSJ9-affect the proficiency of excision repair (15). Many of the yeast excision repair genes are multifunctional, and they play diverse roles in other cellular processes. For example, RAD3 is essential for viability (17,18) and RAD) and RADIO finction in mitotic recombination (19,20). The RAD3 and RAD10 proteins have been purified and characterized in our laboratory. RAD3 is a single-stranded DNA-dependent ATPase, and it possesses DNA and DNARNA helicase activities (21-23). Mutational inactivation of RAD3 ATPase/helicase activities renders cells UV-sensitive and defective in excision repair but has no effect on viability (24). The RAD10 protein binds preferentially to single-stranded DNA and it promotes the renaturation of complementary strands of DNA (25).The yeast RAD3, RADIO, and RADi4 genes are homologs of human XPDC/ERCC2, ERCCJ, and XPAC genes, respectively (6-9, 16). Since none of the known yeast genes showed homology to the human XPBC/ERCC3 gene (desa XPBC in this paper), we cloned the yeast homolog by using XPBC cDNA as a hybridization probe. The yeast homolog has been cloned independently as a suppressor of blocked translation initiation due to the presence of a stem-loop structure in the 5' untranslated region of HIS4 mRNA, and those workers have named the gene SSL2 (26