Transcription-coupled repair in RNA polymerase I-transcribed genes of yeast

Abstract

Nucleotide excision repair (NER) of UV-induced cyclobutane pyrimidine dimers (CPDs) was measured in the individual strands of transcriptionally active and inactive ribosomal genes of yeast. Ribosomal genes (rDNA) are present in multiple copies, but only a fraction of them is actively transcribed. Restriction enzyme digestion was used to specifically release the transcriptionally active fraction from yeast nuclei, and selective psoralen crosslinking was used to distinguish between active and inactive rDNA chromatin. Removal of CPDs was followed in both rDNA populations, and the data clearly show that strand-specific repair occurs in transcriptionally active rDNA while being absent in the inactive rDNA fraction. Thus, transcription-coupled repair occurs in RNA polymerase I-transcribed genes in yeast. Moreover, the nontranscribed strand of active rDNA is repaired faster than either strand of inactive rDNA, implying that NER has preferred access to the active, non-nucleosomal rDNA chromatin. Finally, restriction enzyme accessibility to active rDNA varies during NER, suggesting that there is a change in ribosomal gene chromatin structure during or soon after CPD removal. N ucleotide excision repair (NER) removes different types of lesions from DNA, including bulky adducts caused by chemicals, interstrand or intrastrand crosslinks, and the UV photoproducts cis-syn cyclobutane pyrimidine dimer (CPD) and pyrimidine (6-4) pyrimidone (1). If DNA lesions are not removed, mutations can occur after translesion replication (2). It is now well established that many transcriptionally active genes are repaired faster than inactive DNA (3, 4). Furthermore, preferential removal of CPDs from active genes is caused mainly by an increased rate of repair of the transcribed strand (TS). This transcription-coupled repair (TCR) was first discovered in mammalian cells (5), then in Escherichia coli (6) and yeast (7). Elongation by RNA polymerase II (pol II) is required for TCR (8, 9), and it is thought that only pol II-transcribed genes are subject to TCR (10).RNA polymerase I (pol I) transcribes ribosomal genes (rDNA) at a very high rate. The rDNA is localized in the nucleolus, which is a dense chromatin region composed of rDNA, pol I, rRNA, assembling ribosomes, and proteins involved in cell-cycle regulation (11,12). It is known that mammalian cells repair rDNA damaged by UV radiation (13) and chemicals (14-16). However, in rodent and human cells, CPDs are less efficiently repaired in rDNA than in either total genomic DNA or pol II-transcribed genes (16)(17)(18)(19)(20). Moreover, DNA repair does not exhibit strand bias in the rDNA of these cells (17,19). To the contrary, CPDs are rapidly removed from both strands of total rDNA in yeast (21,22). In addition, a strand bias during repair of total rDNA was observed in rad7 and rad16 mutants, even though strand-specific repair was not observed in total rDNA of wild-type cells (21).Ribosomal genes are present in multiple copies organized in long tandem repeats (11), and in most cells only a f...

RNA Polymerase I Transcription Factors in Active Yeast rRNA Gene Promoters Enhance UV Damage Formation and Inhibit Repair

“…Compared with conventional nuclease footprinting (5), the UV photofootprinting and repair approach has two major advantages: First, UV irradiation and repair is done in living cells. Second, it leaves the DNA intact and the transcribed genes remain nucleosome free, which allows the fractionation of active and inactive genes (10,17,30) and promoters (Fig. 3).…”

Repair-Independent Chromatin Assembly onto Active Ribosomal Genes in Yeast after UV Irradiation

“…Yeast cells (ϳ2 ϫ 10 9 ) were collected, washed with ice-cold phosphate-buffered saline, suspended in 1.5 ml of nuclei isolation buffer (NIB; 50 mM morpholinepropanesulfonic acid [MOPS], pH 8.0, 150 mM potassium acetate, 2 mM MgCl2, 17% glycerol, 0.5 mM spermine, and 0.15 mM spermidine) and transferred to 15-ml polypropylene tubes containing 1.5 ml of glass beads (425 to 600 m; Sigma). Cell disruption and nuclei preparation were done as described previously (7). Nuclei were suspended in 0.5 ml of restriction enzyme buffers (New England BioLabs) and digestions were carried out according to manufacturers' recommendations.…”

“…This transcription-coupled repair process (or TCR) has been thought to require elongating RNA polymerase II (5, 27). However, recently TCR was found in the active fraction of ribosomal genes (rDNA) in yeast wild-type (wt) cells (7,35), which are transcribed at a very high rate by RNA polymerase I. Furthermore, strand-specific repair was observed in total rDNA of rad7⌬, rad16⌬, and rad4⌬ Saccharomyces cerevisiae strains (61).…”

Mammalian Transcription-Coupled Excision Repair

“…A number of functions have been allocated to CSB, including a role of CSB in transcription elongation, nucleosome assembly and histone tail binding, chromatin maintenance and remodeling, and strand annealing and exchange (Selby and Sancar 1997b;Citterio et al 2000;Muftuoglu et al 2006). In addition to its role in coupling RNA-PII arrest and TC-NER, CSB has been reported to localize in mammalian nucleoli as a component of RNAPI transcription machinery together with TFIIH and XPG, and TC-NER of RNAPI transcribed genes were reported to occur in yeast (Bradsher et al 2002;Conconi et al 2002). Furthermore, CSB was shown to localize in mitochondria (mt) and has been implicated in the repair of stress/aging-induced lesions in mtDNA and in apoptosis-mediated loss of subcutaneous fat in mice (Aamann et al 2010;Kamenisch et al 2010).…”