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INSaccharomyces cerevisiae, of the many genes required for excision repair of ultraviolet-damaged DNA, only RAD1 and RAD10 also function in genetic recombination1. Complex formation between the RAD1 and RAD10 gene products1 activates an endo-nucleolytic function that nicks single-stranded DNA2,3 and negatively supercoiled double-stranded DNA2. To characterize the recombination role of the proteins Radl and Radl0, we have investigated their interaction with the Holliday junction, a four-stranded structure that results from single-stranded crossover between two duplex DNA molecules and whose resolution is obligatory for the generation of mature recombinants. We show that Radl binds specifically to a Holliday junction and, in the presence of magnesium, catalyses the endonucleolytic cleavage of the junction. Junction cleavage by Radl proceeds sufficiently without Radl0, thus identifying Radl as the catalytic subunit of Radl/Radl0 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 pigmento-sum (XP) in humans1,2. TheRAD2 gene is one of at least seven genes indispensable for excision repair in the yeast Saccharomyces cerevisiae2, and its encoded protein shares remarkable homology with the XP group-G gene product3. 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 doublestranded 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.

XERODERMA pigmentosum (XP), a genetically heterogeneous human disease, results from a defect in nucleotide excision repair of ultraviolet-damaged DNA. XP patients are extremely sensitive to sunlight and suffer from a high incidence of skin cancers. Cell fusion studies have identified seven XP complementation groups, A–G1–3. Group D is of particular interest as mutations in this gene can also cause Cockayne's syndrome and trichothiodystrophy4. The XPD gene was initially named ERCC2 (excision repair cross complementing) as it was cloned using human DNA to complement the ultraviolet sensitivity of a rodent cell line5. We have purified the XPD protein to near homogeneity and show that it possesses single-stranded DNA-dependent ATPase and DNA helicase activities. We tested whether XPD can substitute for its yeast counterpart RAD3, which is essential for excision repair and for cell viability6. Expression of the XPD gene in Saccharomyces cerevisiae can complement the lethality defect of a mutation in the RAD3 gene6, suggesting that XPD is an essential gene in humans.

XERODERMA pigmentosum (XP), a human autosomal recessive disorder, is characterized by extreme sensitivity to sunlight and high incidence of skin cancers. XP cells are defective in the incision step of excision repair of DNA damaged by ultraviolet light. Cell fusion studies have defined seven XP complementation groups, XP-A to XP-G (refs 1,2). Similar genetic complexity of excision repair is observed in the yeast Saccharomyces cerevisiae. Mutations in any one of five yeast genes, RAD1, RAD2, RAD3, RAD4, and RAD10, cause a total defect in incision and an extreme sensitivity to ultraviolet light3. Here we report the characterization of the yeast RAD14 gene. The available radl4 point mutant is only moderately ultraviolet-sensitive, and it performs a substantial amount of incision of damaged DNA4,5. Our studies with the radl4 deletion (A) mutation indicate an absolute requirement of RAD14 in incision. RAD14 encodes a highly hydrophilic protein of 247 amino acids containing zinc-finger motifs, and it is similar to the protein encoded by the human XPAC gene that complements XP group A cell lines6.

Almost all DNA polymerases show a strong preference for incorporating the nucleotide that forms the correct Watson-Crick base pair with the template base. In addition, the catalytic efficiencies with which any given polymerase forms the four possible correct base pairs are roughly the same. Human DNA polymerase-iota (hPoliota), a member of the Y family of DNA polymerases, is an exception to these rules. hPoliota incorporates the correct nucleotide opposite a template adenine with a several hundred to several thousand fold greater efficiency than it incorporates the correct nucleotide opposite a template thymine, whereas its efficiency for correct nucleotide incorporation opposite a template guanine or cytosine is intermediate between these two extremes. Here we present the crystal structure of hPoliota bound to a template primer and an incoming nucleotide. The structure reveals a polymerase that is 'specialized' for Hoogsteen base-pairing, whereby the templating base is driven to the syn conformation. Hoogsteen base-pairing offers a basis for the varied efficiencies and fidelities of hPoliota opposite different template bases, and it provides an elegant mechanism for promoting replication through minor-groove purine adducts that interfere with replication.

THE RAD10 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of ultraviolet-damaged DNA (refs 1, 2), and it functions in mitotic recombination3. RAD10 has homology to the human excision repair gene ERCC-1 (ref.4). Here we describe the purification of the protein encoded by RAD10 and show that it is a DNA-binding protein with a strong preference for single-stranded DNA. We also show that RAD1O promotes the renaturation of complementary DNA strands.

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.

THE RAD3 gene of Saccharomyces cerevisiae is required for excision repair of ultraviolet-damaged DNA and is essential for cell viability1. The RAD3-encoded protein shares a high degree of homology with the human ERCC2(XPD) gene product2. Mutations in XPD, besides causing the cancer-prone syndrome xeroderma pigmentosum, can also result in Cockayne's syndrome and trichothiodystrophy3. To investigate the role of RAD3 in viability, we examine 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 therad3-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.

We have solved the crystal structure of human DNA polymerase-ι (hPolι) bound to a template primer and incoming nucleotide, and shown that hPolι uses Hoogsteen base-pairing for normal DNA synthesis1. Jimin Wang questions the space group we used and claims that Hoogsteen base-pairing is not supported by our electron-density maps2. We show here that the issue of space group does not affect our conclusions1 and that our electron-density maps are clear in indicating that Hoogsteen and not Watson–Crick base-pairing occurs in this situation.