Repair of rDNA in Saccharomyces Cerevisiae: RAD4-Independent Strand-Specific Nucleotide Excision Repair of RNA Polymerase I Transcribed Genes

Richard A. Verhage 0 1 Pieter van de Putte 0 1 Jaap Brouwer 0 1 0 PO Box 9502, 2300 RA Leiden, The Netherlands 1 Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University Removal of UV-induced pyrimidine dimers from the individual strands of the rDNA locus in Saccharomyces cerevisiae was studied. Yeast rDNA, that is transcribed by RNA polymerase I (RNA pol I), is repaired efficiently, slightly strand-specific and independently of RAD26, which has been implicated in transcription-coupled repair of the RNA pol II transcribed RPB2 gene. No repair of rDNA is observed in rad1, 2, 3 and 14 mutants, demonstrating that dimer removal from this highly repetitive DNA is accomplished by nucleotide excision repair (NER). In rad7 and rad16 mutants, which are specifically deficient in repair of non-transcribed DNA, there is a clear preferential repair of the transcribed strand of rDNA, indicating that strand-specific and therefore probably transcription-coupled repair of RNA pol I transcribed genes does exist in yeast. Unexpectedly, the transcribed but not the non-transcribed strand of rDNA can be repaired in rad4 mutants, which seem otherwise completely NER-deficient. - Cyclobutane pyrimidine dimers induced in DNA by irradiation with UV-light can be removed by the nucleotide excision repair (NER) system to maintain the genetic integrity (reviewed in13). Removal of dimers from DNA is heterogeneous throughout the genome (4,5) because dimers can be a substrate for either of two subpathways of NER: transcription-coupled and global genome repair (6). Transcription-coupled repair is a very efficient process in which lesion-stalled RNA polymerase II (RNA pol II) molecules may act as a condensation site for the assembly of repair complexes (79). Specific gene products might enhance the efficiency of this process. In Escherichia coli, a protein called TRCF (transcription repair coupling factor) couples the NER enzymes to a lesion-stalled RNA polymerase (10). Based on in vitro studies, the following model for transcription-coupled repair in E.coli has been proposed (10): TRCF releases the stalled polymerase together with the transcript, binds the NER protein UvrA, thereby recruiting the NER proteins to lesions that interfere * To whom correspondence should be addressed with transcription. Subsequently these lesions are removed by the action of the Uvr enzymes. In mammalian cells the genes complementing the hereditary recessive disorder Cockayne syndrome groups A and B are involved in transcription-coupled repair (1113), while in S.cerevisiae the homolog of the Cockayne syndrome B gene, RAD26, is implicated in this process (14). It is still unknown whether these genes encode coupling factors analogous to TRCF in E.coli, or are involved in transcription-coupled repair in a different way. Non-transcribed DNA obviously can not be a substrate for transcription-coupled repair. Nevertheless this DNA is repaired by NER enzymes, although slower than transcribed strands (4), in a process referred to as global genome repair. Specific genes have been shown to be essential for global genome repair. Notably, in human xeroderma pigmentosum group C (XP-C) cells, non-transcribed DNA is not repaired while transcribed strands of active DNA are repaired efficiently (15,16). In yeast the RAD7 and RAD16 genes are essential for repair of non-transcribed DNA (17,18). In rad7 and rad16 mutants the transcribed strand of active genes is repaired as efficiently as in RAD+ cells, showing that transcriptioncoupled repair is not hampered in these mutants (18). The actual repair process is conducted by a complex of enzymes called repairosome (19), which contains most proteins that are essential for NER known so far. Most likely this multiprotein complex performs the incisions and subsequent steps in the same manner for both DNA strands. Possibly the repairosome is unable to remove dimers in DNA that is condensed into chromatin, and therefore is dependent on either global genome repair factors or transcription to be able to operate in vivo (6). Transcriptioncoupled repair has been demonstrated in eukaryotes for genes transcribed by RNA polymerase II (RNA pol II) (2023), but not for genes transcribed by RNA pol I (24,25). Here we investigate the repair of ribosomal DNA (rDNA) in yeast, to find out whether RNA pol I transcribed DNA is repaired in a similar way as the genes transcribed by RNA pol II that have been studied so far. rDNA genes are highly repetitive in all organisms, with yeast having 100200 copies (reviewed in 26,27). Two structurally and transcriptionally different subclasses of rDNA exist: some of the copies are inactive and packed in nucleosomal arrays which are not accessible for psoralen crosslinking while the other copies are transcriptionally active and in an open non-nucleosomal chromatin conformation that can be crosslinked by psoralen (28,29). Removal of dimers from rDNA was virtually absent in hamster cells and inefficient in human cells (24,25). It was speculated that removal of dimers from the highly repetitive rDNA cluster could be due to recombination instead of NER (24), but subsequently it was shown that in XP-C and CS-B cells which are impaired in NER, repair of rDNA was inhibited (30). Repair of mammalian rDNA appeared to be not strand-specific (not transcription-coupled) and less efficient than repair of the genome overall (24,25). We have studied removal of dimers from the rDNA cluster of yeast in repair proficient (RAD+) cells and in various rad mutants that are disturbed in specific subpathways of NER. Our results reveal marked differences between repair of rDNA in yeast compared to results described for mammalian cells, as well as differences in repair of rDNA and genes that are transcribed by RNA pol II. The data also have implications for the function of Rad4p in NER, and possibly for its presumed human homolog, XPC. MATERIALS AND METHODS All general procedures including DNA purification, restriction enzyme digestion, cloning and gel electrophoresis were performed according to standard procedures (31). Plasmids were propagated in E.coli strain JM101 under appropriate antibiotic selection. Yeast strains and media The yeast strains used for this study are listed in Table 1. All strains were kept on selective YNB (0.67% yeast nitrogen base, 2% glucose, 2% bacto agar) supplemented with the appropriate markers. Cells were grown in complete medium (YEPD: 1% yeast extract, 2% bacto peptone, 2% glucose) at 28 C under vigorous shaking conditions. Construction of disruption mutants Yeast cells were transformed by electroporation (2250 V/cm, 250 m F, 200 W ). Cells were plated on YNB with the necessary amino acids and incubated at 28 C for 25 days. Disruption of the RAD4 gene was accomplished by transformation of XbaI-digested pDG38 (gift of D. Gietz). Disruptions of the RAD14 gene were obtained by transformation of SacI/NcoI-digested pBM190 (gif (...truncated)