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Raymond Waters

Publications and source records attributed to Raymond Waters.

10 recordsLinked to original sources
PubMed2006

Distinct functions of the ubiquitin-proteasome pathway influence nucleotide excision repair.

The Rad23/Rad4 nucleotide excision repair (NER) protein complex functions at an early stage of the NER reaction, possibly promoting the recognition of damaged DNA. Here we show that Rad4 protein is ubiquitinated and degraded in response to ultraviolet (UV) radiation, and identify a novel cullin-based E3 ubiquitin ligase required for this process. We also show that this novel ubiquitin ligase is required for optimal NER. Our results demonstrate that optimal NER correlates with the ubiquitination of Rad4 following UV radiation, but not its subsequent degradation. Furthermore, we show that the ubiquitin-proteasome pathway (UPP) regulates NER via two distinct mechanisms. The first occurs independently of de novo protein synthesis, and requires Rad23 and a nonproteolytic function of the 19S regulatory complex of the 26S proteasome. The second requires de novo protein synthesis, and relies on the activity of the newly identified E3 ubiquitin ligase. These studies reveal that, following UV radiation, NER is mediated by nonproteolytic activities of the UPP, via the ubiquitin-like domain of Rad23 and UV radiation-induced ubiquitination of Rad4.

DNA Repair↗
PubMed2005

Histone acetylation, chromatin remodelling and nucleotide excision repair: hint from the study on MFA2 in Saccharomyces cerevisiae.

Nucleotide excision repair (NER) is a sophisticated repair pathway that the cell utilizes to remove a broad range of DNA damage to help maintain the functional integrity of the genome. In the context of DNA packaged into chromosomes it is clear that the NER machinery does not repair all regions with equal efficiency. Recently, we found after UV that histone acetylation and chromatin remodelling were activated. UV irradiation triggers genome-wide histone hyperacetylation at both histone H3 and H4. However, in nucleosomes at the repressed MFA2 promoter only histone H3, but not histone H4, is hyperacetylated following UV. This Gcn5p-mediated histone H3 hyperacetylation enables efficient NER at MFA2. Chromatin in this promoter also becomes more accessible after UV. This is not dependent on Gcn5p, yet it is partially dependent on Swi2p. In later repair times both events gradually return to the pre-UV state. The post-UV histone modifications and chromatin remodelling at the repressed MFA2 promoter do not activate MFA2 transcription, nor do they require damage recognition by Rad4p or Rad14p. These experiments indicate early events are triggered in chromatin in response to UV treatment, and they are likely needed for efficient NER.

Acetylation↗
PubMed2005

Preface.

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Chromatin↗
PubMed2005

Histone acetylation, chromatin remodelling, transcription and nucleotide excision repair in S. cerevisiae: studies with two model genes.

We describe the technology and two model systems in yeast designed to study nucleotide excision repair (NER) in relation to transcription and chromatin modifications. We employed the MFA2 and MET16 genes as models. How transcription-coupled (TCR) and global genome repair (GGR) operate at the transcriptionally active and/or repressed S. cerevisiae MFA2 locus, and how this relates to nucleosome positioning are considered. We discuss the role of the Gcn5p histone acetyltransferase, also associated with MFA2's transcriptional activation, in facilitating efficient NER at the transcriptionally active and inactive genes. The effect of Gcn5p's absence in reducing NER was local and UV stimulates Gcn5p-mediated histone acetylation at the repressed MFA2 promoter. After UV irradiation Swi2p is partly responsible for facilitating access to restriction of DNA in the cores of the nucleosomes at the MFA2 promoter. The data suggest similarities between chromatin remodelling for NER and transcription, yet differences must exist to ensure this gene remains repressed in alpha cells during NER. For MET16, we consider experiments examining chromatin structure, transcription and repair in wild type and cbf1Delta cells under repressing or derepressing conditions. Cbf1p is a sequence specific DNA binding protein required for MET16 chromatin remodelling and transcription.

Acetylation↗
PubMed2005

UV irradiation stimulates histone acetylation and chromatin remodeling at a repressed yeast locus.

Chromatin immunoprecipitation with anti-acetyl histone H3 (K9 and K14) and anti-acetyl histone H4 (K5, K8, K12, and K16) antibodies shows that Lys-9 and/or Lys-14 of histone H3, but not the relevant sites of histone H4 in nucleosomes at the repressed MFA2 promoter, are hyperacetylated after UV irradiation. This level of histone hyperacetylation diminishes gradually as repair proceeds. Accompanying this, chromatin in the promoter becomes more accessible to restriction enzymes after UV irradiation and returns to the pre-UV state gradually. UV-related histone hyperacetylation and chromatin remodeling in the MFA2 promoter depend on Gcn5p and partially on Swi2p, respectively. Deletion of GCN5, but not of SWI2, impairs repair of DNA damage in the MFA2 promoter. The post-UV histone modifications and chromatin remodeling at the repressed MFA2 promoter do not activate MFA2 transcriptionally, nor do they require damage recognition by Rad4p or Rad14p. Furthermore, we show that UV irradiation triggers genome-wide histone hyperacetylation at both histone H3 and H4. These experiments indicate that chromatin at a yeast repressed locus undergoes active change after UV radiation treatment and that failure to achieve histone H3 hyperacetylation impairs the repair of DNA damage.

Acetylation↗
PubMed2004

The yeast Rad7/Rad16/Abf1 complex generates superhelical torsion in DNA that is required for nucleotide excision repair.

Nucleotide excision repair (NER) in eukaryotes removes DNA base damage as an oligonucleotide in a complex series of reactions. The nature of the dual incision reactions on either side of the damaged base has been extensively investigated. However, the precise mechanism of cleavage of the phosphodiester backbone of the DNA by the NER endonucleases and how this relates to removal of the damage-containing oligonucleotide during the excision process has not been determined. We previously isolated a stable heterotrimeric complex of Rad7/Rad16/Abf1 from yeast which functions in the conserved global genome repair (GGR) pathway. GGR removes lesions from DNA that is not actively transcribing. We have shown previously that the Rad7/Rad16/Abf1 heterotrimer is required to observe DNA repair synthesis and oligonucleotide excision during in vitro NER, but not needed to detect NER-dependent incision in such reactions. Here we report that this protein complex generates superhelicity in DNA through the catalytic activity of the Rad16 component. The torsion generated in the DNA by this complex is necessary to remove the damage-containing oligonucleotide during NER--a process referred to as excision. We conclude that in yeast the molecular mechanism of NER includes the generation of superhelical torsion in DNA.

Adenosine Triphosphatases↗
PubMed2003

Transcription, nucleosome positioning and protein binding modulate nucleotide excision repair of the Saccharomyces cerevisiae MET17 promoter.

We have assessed how transcription, chromatin structure and protein binding modulate nucleotide excision repair in the upstream regulatory region and early coding region of the endogenous Saccharomyces cerevisiae gene MET17. Removal of UV-induced cyclobutane pyrimidine dimers was measured from these regions, in which transcription and chromatin structure could be regulated independently of each other. Distinct repair trends were apparent depending on transcriptional state. When transcription was repressed nucleosome positioning and protein binding as determined by chromatin immunoprecipitation and quantitative real-time PCR, were significant factors. Nucleosome positioning and/or protein binding effects were most apparent on the strand that becomes transcribed, with repair occurring fastest in a nucleosome free region but being retarded where regulatory proteins bound within this region. When transcription was derepressed the rate of repair increased on both strands in a region beginning 200 bp upstream of the TATA box and extending downstream into the coding region. This effect overrode the influences of nucleosome positioning and protein binding.

DNA↗
PubMed2002

Photoreactivation of UV-induced cyclobutane pyrimidine dimers in the MFA2 gene of Saccharomyces cerevisiae.

Photoreactive repair (PR) of cyclobutane pyrimidine dimers (CPDs) was mapped at nucleotide resolution in nucleotide excision repair (NER) proficient and deficient strains for the transcriptionally active and inactive MFA2 gene. Enhanced PR in the control region occurred in areas where no nucleosomes were present, particularly linker regions in the alpha mating type. The presence of excision plus transcriptional activation alleviated this preference, causing repair in the regions that were linker and core in the alpha mating type to be the same in this strain. Transcription had no effect on photoreactive repair in transcribed and downstream regions of MFA2, where similar rates were observed for specific CPDs in both strands. The presence of positioned nucleosomes in alpha mating types revealed slow repair in the nucleosome core, with faster repair occurring at the 3' and 5' edges. These data support the evidence that CPDs are repaired quicker in nucleosome-free regions and at edges of nucleosomes. CPDs in the linker regions are repaired more efficiently in the transcriptionally inactive strains, suggesting that nucleosome movement associated with transcription of MFA2 hampers PR irrespective of the strand. Proficient NER influenced PR in the TATA and Mcm1 binding sites by enhancing it, particularly when transcription was activated.

Autoradiography↗
PubMed2002

The Saccharomyces cerevisiae histone acetyltransferase Gcn5 has a role in the photoreactivation and nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers in the MFA2 gene.

How DNA repair enzymes or complexes gain access to chromatin is still not understood. Here, we have studied the role of the S. cerevisiae histone acetyltransferase Gcn5 in photoreactivation (PR) and nucleotide excision repair (NER) at the level of the genome, the MFA2 and RPB2 genes, and at specific nucleotides within MFA2. The deletion of GCN5 markedly reduced the PR and NER of UV-induced cyclobutane pyrimidine dimers in MFA2 but much less so in RPB2, whereas no detectable defect was seen for repair of the genome overall. In Delta(gcn5), the MFA2 mRNA level is reduced by fourfold, while transcription from RPB2 is reduced only to 80 %. These changes in transcription correlate with the changes in NER and PR found in the Delta(gcn5) mutant. However, changes in MFA2 transcription cannot account for the decrease in NER in the non-transcribed strand and the control region of MFA2 where global genome repair (GGR) operates. We conclude that the histone acetyltransferase Gcn5 influences PR and NER at MFA2 in both its transcribed and non-transcribed DNA, yet it has little effect on these processes for most of the yeast genome. As a result, we speculate that histone acetylation allows efficient access of the repair machinery to chromosomal DNA damages either indirectly via influencing transcription or directly via modifying chromatin structure irrespective of transcription.

Acetyltransferases↗