Differential repair of UV damage in<i>Saccharomyces cerevisiae</i>

Terleth, Carrol; Sluis, C.A. van; Putte, Pieter van de

doi:10.1093/nar/17.12.4433

Cited by 117 publications

(41 citation statements)

References 24 publications

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“…The coupling of transcription to repair helps to prevent such a catastrophe. Such coupling, although not universal for all adducts (22) or all genes (23), has been documented in vivo in several systems (4)(5)(6)(7)23), and it also has been inferred from studies showing selective mutagenesis (24,25 Partial purification of a coupling factor that enhances repair oftemplate DNA when added to a defined transcription-repair system. The defined system consisted ofpDR3274 irradiated with 225 Jm-2 of UV as the template-substrate for purified RNA Pol, UvrA, UvrB, UvrC, UvrD, pol I, and DNA ligase.…”

Section: Resultsmentioning

confidence: 99%

“…Consequently it was found that in both CHO and human cells, the template strand of the actively transcribed DHFR gene was repaired 2-to 10-fold more efficiently than the complementary strand, which was repaired at a rate comparable to that of nontranscribed regions (4). These observations, originally made in mammalian cells, have now been extended to other organisms including yeast (5,6) and Escherichia coli (7).…”

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confidence: 94%

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Gene- and strand-specific repair in vitro: partial purification of a transcription-repair coupling factor.

Selby

Sancar

1991

Proc. Natl. Acad. Sci. U.S.A.

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In eukaryotic and prokaryotic cells, actively transcribed genes and, in some instances, the template strand of these genes have been found to be repaired 2-10 times more rapidly than nontranscribed genes or the coding strand of transcribed genes. We demonstrate here gene-and template strand-specific repair synthesis in vitro by using an Eschenchia col cell-free extract and a plasmid carrying a gene with the strong tac promoter. Strand-specific repair of UV, 4'-hydroxymethyl-4,5',8-trimethylpsoralen, and cis-dicholorodiammine platinum(II) damage was dependent upon transcription and a functional nucleotide excision repair system and was stimulated by 6% (wt/vol) polyethylene glycol. A defined system consisting of the transcription and repair proteins in highly purified form did not perform strand-specific repair; however, active fractions of extract conferred strand specificity to the defied system. Transcription-repair coupling activity was partially purified from extract by successive DEAEagarose and gel filtration chromatography. The coupling factor is heat-labile, with an estimated Mr of 100,000.damaged DNA but does not take part in the DNA incision, which is carried out by UvrB-UvrC.] This study yielded a paradoxical result: RNA Pol stalled at a thymine dimer (TOT) in the template strand prevented access of (A)BC excinuclease (which incises the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to the photodimer) to the lesion. Thus, a TOT in the complementary (coding) strand (which did not block transcription) was repaired 2-to 4-fold more efficiently than a TOT in the template strand (8). This result suggests that "transcription-repair coupling" (9) must accomplish two tasks: direct the repair enzyme (assembly) to the stalled complex and overcome the steric hindrance caused by the stalled RNA Pol. In this paper we show that a more complete but less-defined in vitro system utilizing E. coli cell-free extract carries out gene-and strand-specific repair. A coupling factor that conferred strand-specific repair function when added to a defined transcription-repair system was partially purified.DNA repair enzymes in general and nucleotide-excision nuclease subunits in particular are not abundant (1, 2), and it would be advantageous if the cell's limited resources were channeled to the most threatening genetic lesions. In this regard, while replication-blocking lesions could be lethal in any location on the chromosome, lesions in genes pose the additional threats of causing deleterious mutations or blocking transcription. It now appears that eukaryotes and prokaryotes use specific targeting mechanisms for some types of lesions to direct nucleotide-excision repair to regions most crucial for survival.The first and perhaps most influential report on the subject by Bohr et al. (3) showed that pyrimidine dimers (PyrOPyr) in the dihydrofolate reductase (DHFR) gene of Chinese Hamster ovary (CHO) cells were repaired about five times more rapidly than those in flanking nontranscribed regions or in...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 94%

Gene- and strand-specific repair in vitro: partial purification of a transcription-repair coupling factor.

Selby

Sancar

1991

Proc. Natl. Acad. Sci. U.S.A.

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“…MAT␣ and HML␣ fragments were generated by digesting genomic DNA with HaeII enzyme, which yields a 3.6-kb MAT␣ fragment and a 4.0-kb HML␣ fragment. The probe was an internal fragment of the Y␣ region of MAT␣, which is strictly ␣-specific (see below) (21). DNA (5 g) was restricted with HaeII and incubated at 37°C for 2 h. The DNA was recovered after phenol-chloroform extraction and resuspended in 10 l of denaturing loading buffer (12.5% Ficoll, 5 mM EDTA, 0.125% bromphenol blue, 0.25 M NaOH).…”

Section: Strains and Plasmids-the Radmentioning

confidence: 99%

The Yeast RAD7 and RAD16 Genes Are Required for Postincision Events during Nucleotide Excision Repair

Reed

You

Friedberg

1998

Journal of Biological Chemistry

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In eukaryotes, nucleotide excision repair (NER) is a complex reaction requiring multiple proteins. In the yeast Saccharomyces cerevisiae, two of these proteins, Rad7 and Rad16, are specifically involved in the removal of lesions from transcriptionally silent regions of the genome in vivo. Extracts prepared from rad7 or rad16 mutant cells are deficient, but not totally defective, in both oligonucleotide excision and repair synthesis of damaged plasmid DNA. We show that these extracts are, however, fully proficient in the incision step of the NER reaction in vitro. Furthermore, using a cdc9 mutant to trap incision intermediates, we demonstrate that rad7 and rad16 mutants are proficient in NER-dependent DNA incision in vivo. A purified protein complex containing both Rad7 and Rad16 proteins complements the oligonucleotide excision and repair synthesis defects in rad7 and rad16 mutant extracts. We conclude that the products of the RAD7 and RAD16 genes are involved in a postincision event(s) during NER in yeast.The yeast RAD7 and RAD16 genes belong to the RAD3 epistasis group of DNA damage-responsive genes (1, 2). This epistasis group includes genes required for nucleotide excision repair (NER) 1 of DNA, a process by which multiple types of base damage are excised from the genome as oligonucleotide fragments, and the resulting single strand gaps are repaired by DNA synthesis (repair synthesis) and ligation (3). In contrast to the extreme sensitivity to UV light (and other DNA-damaging agents) conferred by complete inactivation of NER genes, such as RAD1, RAD2, RAD3, RAD4, RAD10, and RAD14, deletion of the RAD7 or RAD16 gene confers partial UV radiation sensitivity (1, 2).The precise role(s) of the RAD7 and RAD16 gene products in NER is not clear. The proteins are required for NER of transcriptionally repressed loci such as HML␣ and HMRa in vivo (4), and for the nontranscribed (coding) strand of transcriptionally active genes. However, unlike the other RAD genes mentioned above, the RAD7 and RAD16 genes are not required for NER of the transcribed (template) strand of such genes (5). The Rad7 and Rad16 proteins have been shown to stably interact both in vivo and in vitro (6, 9). Rad16 is a member of the SWI2/SNF2 superfamily of proteins, several of which have been shown to be ATPases involved in chromatin remodeling (7). This observation, coupled with the requirement for the RAD7 and RAD16 genes for NER of transcriptionally repressed regions of the genome, has led to the notion that the Rad7 and Rad16 proteins may be subunits of a complex dedicated to the perturbation of chromatin structure in order to facilitate NER of transcriptionally silent regions of the genome and the nontranscribed strand of transcriptionally active genes (5).It has been reported that a reconstituted in vitro system that supports damage-specific incision and oligonucleotide excision from purified plasmid DNA does not require the Rad7 and Rad16 proteins (8). Subsequent studies (9) reported that these proteins actually increase the efficiency of N...

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“…Studies of repair in unique chromosomal DNA sequences did not show a strand bias in repair (7)(8)(9). In contrast, Smerdon and Thoma and colleague (10,11) reported that excision repair of CPDs in a plasmid was rapid for transcribed strands, while nontranscribed sequences were slowly repaired.…”

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confidence: 95%

Preferential repair of cyclobutane pyrimidine dimers in the transcribed strand of a gene in yeast chromosomes and plasmids is dependent on transcription.

Sweder

Hanawalt

1992

Proc. Natl. Acad. Sci. U.S.A.

162

112

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While preferential repair of the transcribed strands within active genes has been demonstrated in organisms as diverse as humans and Escherichia colt, it has not previously been shown to occur in chromosomal genes in the yeast Saccharomyces cerevisiae. We found that repair of cyclobutane pyrimidine dimers in the transcribed strand of the expressed RPB2 gene in the chromosome of a repair-proficient strain is much more rapid than that in the nontranscribed strand. Furthermore, a copy of the RPB2 gene borne on a centromeric ARSI plasmid showed the same strand bias in repair. To investigate the relation of this strand bias to transcription, we studied repair in a yeast strain with the temperature-sensitive mutation, rpbl-1, in the largest subunit of RNA polymerase II. When exponentially growing rpbl-1 cells are shifted to the nonpermissive temperature, they rapidly cease mRNA synthesis. At the permissive temperature, both rpbl-1 and the wild-type, parental cells exhibited rapid, proficient repair in the transcribed strand of chromosomal and plasmid-borne copies of the RPB2 gene. At the nonpermissive temperature, the rate of repair in the transcribed strand in rpbl-1 cells was reduced to that in the nontranscribed strand.These findings establish the dependence of strand bias in repair on transcription by RNA polymerase II in the chromosomes and in plasmids, and they validate the use of plasmids for analysis of the relation of repair to transcription in yeast.Both eukaryotes and prokaryotes carry out excision repair of DNA damage after exposure to UV light (1). The two major classes of lesions produced are 5-5, 6-6 cis-syn cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts, both of which are removed from the DNA. Furthermore, CPD removal has been shown in mammalian cells to be faster in transcriptionally active genes when compared to the genome overall (for recent reviews, see refs. 2 and 3) and more rapid in the transcribed strand in expressed genes than in the nontranscribed strand (4). This preferential repair of the transcribed strand in expressed genes has also been shown for the lac genes in the Escherichia coli chromosome (5) and for the uvrC gene in vitro (6). Thus, it seems likely that preferential DNA repair of active genes is a conserved pathway for nucleotide excision repair in both prokaryotes and eukaryotes. However, preferential DNA repair of the transcribed strand of an active chromosomal gene has not yet been shown in the unicellular eukaryote Saccharomyces cerevisiae.Recent reports of transcription-associated nucleotide excision repair in yeast are seemingly inconsistent. Studies of repair in unique chromosomal DNA sequences did not show a strand bias in repair (7-9). In contrast, Smerdon and Thoma and colleague (10,11) reported that excision repair of CPDs in a plasmid was rapid for transcribed strands, while nontranscribed sequences were slowly repaired. To resolve these differences we have compared the repair rates in the same expressed sequence in a plasmid...

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Differential repair of UV damage inSaccharomyces cerevisiae

Cited by 117 publications

References 24 publications

Gene- and strand-specific repair in vitro: partial purification of a transcription-repair coupling factor.

Gene- and strand-specific repair in vitro: partial purification of a transcription-repair coupling factor.

The Yeast RAD7 and RAD16 Genes Are Required for Postincision Events during Nucleotide Excision Repair

Preferential repair of cyclobutane pyrimidine dimers in the transcribed strand of a gene in yeast chromosomes and plasmids is dependent on transcription.

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