radS (rev2) mutants of Saccharomyces cerevisiae are sensitive to UV light and other DNA-damaging agents, and RADS is in the RAD6 epistasis group of DNA repair genes. To unambiguously define the function of R4DS, we have cloned the RAD5 gene, determined the effects of the radS deletion mutation on DNA repair, DNA damage-induced mutagenesis, and other cellular processes, and analyzed the sequence of RAD5-encoded protein. Our genetic studies indicate that R4DS functions primarily with R4D18 in error-free postreplication repair. We also show that R4DS affects the rate of instability of poly(GT) repeat sequences. Genomic poly(GT) sequences normally change length at a rate of about 10-4; this rate is approximately 10-fold lower in the radS deletion mutant than in the corresponding isogenic wild-type strain. RADS encodes a protein of 1,169 amino acids of M, 134,000, and it contains several interesting sequence motifs. All seven conserved domains found associated with DNA helicases are present in RAD5. RAD5 also contains a cysteine-rich sequence motif that resembles the corresponding sequences found in 11 other proteins, including those encoded by the DNA repair gene RA4D8 and the RAGI gene required for immunoglobin gene arrangement. A leucine zipper motif preceded by a basic region is also present in RAD5. The cysteine-rich region may coordinate the binding of zinc; this region and the basic segment might constitute distinct DNA-binding domains in RAD5. Possible roles of RAD5 putative ATPase/DNA helicase activity in DNA repair and in the maintenance of wild-type rates of instability of simple repetitive sequences are discussed.DNA containing pyrimidine dimers and other types of UV damage is a poor template for the replication machinery. Since DNA polymerases cannot copy past such damage, a gap ensues in the newly synthesized strand across from the damage site. This gap can be filled in by several different postreplication repair processes. Genetic evidence from Saccharomyces cerevisiae suggests that the majority of gaps are filled in an error-free manner in which information for gap filling is obtained from the undamaged strand in the sister duplex (45). Either recombinational or nonrecombinational mechanisms may be utilized. In recombination, the strand from the sister duplex fills in the gap; a nonrecombinational mechanism might entail a copy choice type of DNA synthesis in which blocked DNA polymerase switches from the damaged template, carries out translesion DNA synthesis by copying the undamaged strand in the sister duplex, and then switches back to the damaged template after clearing the lesion. Under certain circumstances, gap filling can occur by mutagenic bypass of the lesion.To elucidate the mechanisms employed by eukaryotic cells to fill in the gap across from the lesion, we have been studying the genes that function in nonmutagenic and mutagenic postreplication repair pathways in S. cerevisiae. Several of these genes have been cloned, and in some cases, their protein products have been purified and their b...

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.

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. 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 light. Here we report the characterization of the yeast RAD14 gene. The available rad14 point mutant is only moderately ultraviolet-sensitive, and it performs a substantial amount of incision of damaged DNA. Our studies with the rad14 deletion (delta) 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 lines.

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