Synergistic interaction between yeast nucleotide excision repair factors NEF2 and NEF4 in the binding of ultraviolet-damaged DNA.

Saccharomyces cerevisiae RAD4, RAD7, RAD16, and RAD23 genes function in the nucleotide excision repair (NER) of ultraviolet light (UV)-damaged DNA. Previous biochemical studies have shown that the Rad4 and Rad23 proteins are associated in a stoichiometric complex named NEF2, and the Rad7 and Rad16 proteins form another stoichiometric complex named NEF4. While NEF2 is indispensable for the incision of UV-damaged DNA in the in vitro reconstituted system, NEF4 stimulates the incision reaction. Both NEF2 and NEF4 bind UV-damaged DNA, which raises the intriguing possibility that these two complexes cooperate to achieve the high degree of specificity for DNA damage demarcation required for nucleotide excision repair in vivo. Consistent with this hypothesis, we find that NEF2 and NEF4 bind in a synergistic fashion to UV-damaged DNA in a reaction that is dependent on ATP. We also purify the Rad7 protein and show that it binds DNA but has no preference for UV-damaged DNA. Rad7 physically interacts with NEF2, suggesting a role for Rad7 in linking NEF2 with NEF4.     

Complex formation with damage recognition protein Rad14 is essential for Saccharomyces cerevisiae Rad1-Rad10 nuclease to perform its function in nucleotide excision repair in vivo

Nucleotide excision repair (NER) in eukaryotes requires the assembly of a large number of protein factors at the lesion site which then coordinate the dual incision of the damaged DNA strand. However, the manner by which the different protein factors are assembled at the lesion site has remained unclear. Previously, we have shown that in the yeast Saccharomyces cerevisiae, NER proteins exist as components of different protein subassemblies: the Rad1-Rad10 nuclease, for example, forms a tight complex with the damage recognition protein Rad14, and the complex of Rad1-Rad10-Rad14 can be purified intact from yeast cells. As the Rad1-Rad10 nuclease shows no specificity for binding UV lesions in DNA, association with Rad14 could provide an effective means for the targeting of Rad1-Rad10 nuclease to damage sites in vivo. To test the validity of this idea, here we identify two rad1 mutations that render yeast cells as UV sensitive as the rad1Delta mutation but which have no effect on the recombination function of Rad1. From our genetic and biochemical studies with these rad1 mutations, we conclude that the ability of Rad1-Rad10 nuclease to associate in a complex with Rad14 is paramount for the targeting of this nuclease to lesion sites in vivo. We discuss the implications of these observations for the means by which the different NER proteins are assembled at the lesion site.     

Damage recognition in nucleotide excision repair of DNA

Nucleotide excision repair (NER) is found throughout nature, in eubacteria, eukaryotes and archaea. In human cells it is the main pathway for the removal of damage caused by UV light, but it also acts on a wide variety of other bulky helix-distorting lesions caused by chemical mutagens. An ongoing challenge is to understand how a site of DNA damage is located during NER and distinguished from non-damaged sites. This article reviews information on damage recognition in mammalian cells and the bacterium Escherichia coli. In mammalian cells the XPC-hHR23B, XPA, RPA and TFIIH factors may all have a role in damage recognition. XPC-hHR23B has the strongest affinity for damaged DNA in some assays, as does the similar budding yeast complex Rad4-Rad23. There is current discussion as to whether XPC or XPA acts first in the repair process to recognise damage or distortions. TFIIH may play a role in distinguishing the damaged strand from the non-damaged one, if translocation along a DNA strand by the TFIIH DNA helicases is interrupted by encountering a lesion. The recognition and incision steps of human NER use 15 to 18 polypeptides, whereas E. coli requires only three proteins to obtain a similar result. Despite this, many remarkable similarities in the NER mechanism have emerged between eukaryotes and bacteria. These include use of a distortion-recognition factor, a strand separating helicase to create an open preincision complex, participation of structure-specific endonucleases and the lack of a need for certain factors when a region containing damage is already sufficiently distorted.     

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.     

Strong functional interactions of TFIIH with XPC and XPG in human DNA nucleotide excision repair, without a preassembled repairosome

In mammalian cells, the core factors involved in the damage recognition and incision steps of DNA nucleotide excision repair are XPA, TFIIH complex, XPC-HR23B, replication protein A (RPA), XPG, and ERCC1-XPF. Many interactions between these components have been detected, using different physical methods, in human cells and for the homologous factors in Saccharomyces cerevisiae. Several human nucleotide excision repair (NER) complexes, including a high-molecular-mass repairosome complex, have been proposed. However, there have been no measurements of activity of any mammalian NER protein complex isolated under native conditions. In order to assess relative strengths of interactions between NER factors, we captured TFIIH from cell extracts with an anti-cdk7 antibody, retaining TFIIH in active form attached to magnetic beads. Coimmunoprecipitation of other NER proteins was then monitored functionally in a reconstituted repair system with purified proteins. We found that all detectable TFIIH in gently prepared human cell extracts was present in the intact nine-subunit form. There was no evidence for a repair complex that contained all of the NER components. At low ionic strength TFIIH could associate with functional amounts of each NER factor except RPA. At physiological ionic strength, TFIIH associated with significant amounts of XPC-HR23B and XPG but not other repair factors. The strongest interaction was between TFIIH and XPC-HR23B, indicating a coupled role of these proteins in early steps of repair. A panel of antibodies was used to estimate that there are on the order of 10(5) molecules of each core NER factor per HeLa cell.     

Functionally distinct nucleosome-free regions in yeast require Rad7 and Rad16 for nucleotide excision repair

In yeast, Rad7 and Rad16 are two proteins required for nucleotide excision repair (NER) of non-transcribed chromatin. They have roles in damage recognition, in the postincision steps of NER, and in ultraviolet-light-dependent histone H3 acetylation. Moreover, Rad16 is an ATP-ase of the SNF2 superfamily and therefore might facilitate chromatin repair by nucleosome remodelling. Here, we used yeast rad7 Delta rad16 Delta mutants and show that Rad7-Rad16 is also required for NER of UV-lesions in three functionally distinct nucleosome-free regions (NFRs), the promoter and 3'-end of the URA3 gene and the ARS1 origin of replication. Moreover, rapid repair of UV-lesions by photolyase confirmed that nucleosomes were absent and that neither UV-damage formation nor rad7 Delta rad16 Delta mutations altered chromatin accessibility in NFRs. The data are consistent with a role of Rad7-Rad16 in damage recognition and processing in absence of nucleosomes. An additional role in nucleosome remodelling is discussed.     

Incision of a 1,3-intrastrand d(GpTpG)-cisplatin adduct by nucleotide excision repair proteins from yeast

The protein machinery responsible for nucleotide excision repair (NER) is highly conserved from yeast to man. NER can be reconstituted with purified proteins, and the incision sites around a defined DNA lesion have been defined to the nucleotide level in a mammalian NER system. Here, we reconstitute NER in yeast whole cell extracts, as well as with partially purified yeast NER components. We show that NER activity can be isolated partly as a large protein complex, and map the sites of nucleotide incision around a cisplatin-induced DNA lesion. Our data indicate that yeast NER proteins excise an oligonucleotide of 23-26 bases containing the DNA lesion (rather than 26-30 bases as in humans), and that the 3' incision occurs around position 17 (rather than at position 9 as in humans).     

Yeast Deubiquitinase Ubp3 Interacts with the 26 S Proteasome to Facilitate Rad4 Degradation

Deubiquitinating enzymes (DUBs) function in a variety of cellular processes by removing ubiquitin moieties from substrates, but their role in DNA repair has not been elucidated. Yeast Rad4-Rad23 heterodimer is responsible for recognizing DNA damage in nucleotide excision repair (NER). Rad4 binds to UV damage directly while Rad23 stabilizes Rad4 from proteasomal degradation. Here, we show that disruption of yeast deubiquitinase UBP3 leads to enhanced UV resistance, increased repair of UV damage and Rad4 levels in rad23Δ cells, and elevated Rad4 stability. A catalytically inactive Ubp3 (Ubp3-C469A), however, is unable to affect NER or Rad4. Consistent with its role in down-regulating Rad4, Ubp3 physically interacts with Rad4 and the proteasome, both in vivo and in vitro, suggesting that Ubp3 associates with the proteasome to facilitate Rad4 degradation and thus suppresses NER.     

The Ino80 chromatin-remodeling complex restores chromatin structure during UV DNA damage repair

Chromatin structure is modulated during deoxyribonucleic acid excision repair, but how this is achieved is unclear. Loss of the yeast Ino80 chromatin-remodeling complex (Ino80-C) moderately sensitizes cells to ultraviolet (UV) light. In this paper, we show that INO80 acts in the same genetic pathway as nucleotide excision repair (NER) and that the Ino80-C contributes to efficient UV photoproduct removal in a region of high nucleosome occupancy. Moreover, Ino80 interacts with the early NER damage recognition complex Rad4-Rad23 and is recruited to chromatin by Rad4 in a UV damage-dependent manner. Using a modified chromatin immunoprecipitation assay, we find that chromatin disruption during UV lesion repair is normal, whereas the restoration of nucleosome structure is defective in ino80 mutant cells. Collectively, our work suggests that Ino80 is recruited to sites of UV lesion repair through interactions with the NER apparatus and is required for the restoration of chromatin structure after repair.     

Requirement of ELC1 for RNA polymerase II polyubiquitylation and degradation in response to DNA damage in Saccharomyces cerevisiae

Treatment of Saccharomyces cerevisiae and human cells with DNA-damaging agents such as UV light or 4-nitroquinoline-1-oxide induces polyubiquitylation of the largest RNA polymerase II (Pol II) subunit, Rpb1, which results in rapid Pol II degradation by the proteasome. Here we identify a novel role for the yeast Elc1 protein in mediating Pol II polyubiquitylation and degradation in DNA-damaged yeast cells and propose the involvement of a ubiquitin ligase, of which Elc1 is a component, in this process. In addition, we present genetic evidence for a possible involvement of Elc1 in Rad7-Rad16-dependent nucleotide excision repair (NER) of lesions from the nontranscribed regions of the genome and suggest a role for Elc1 in increasing the proficiency of repair of nontranscribed DNA, where as a component of the Rad7-Rad16-Elc1 ubiquitin ligase, it would promote the efficient turnover of the NER ensemble from the lesion site in a Rad23-19S proteasomal complex-dependent reaction.     

NER and HR pathways act sequentially to promote UV-C-induced germ cell apoptosis in Caenorhabditis elegans

Ultraviolet (UV) radiation-induced DNA damage evokes a complex network of molecular responses, which culminate in DNA repair, cell cycle arrest and apoptosis. Here, we provide an in-depth characterization of the molecular pathway that mediates UV-C-induced apoptosis of meiotic germ cells in the nematode Caenorhabditis elegans. We show that UV-C-induced DNA lesions are not directly pro-apoptotic. Rather, they must first be recognized and processed by the nucleotide excision repair (NER) pathway. Our data suggest that NER pathway activity transforms some of these lesions into other types of DNA damage, which in turn are recognized and acted upon by the homologous recombination (HR) pathway. HR pathway activity is in turn required for the recruitment of the C. elegans homolog of the yeast Rad9-Hus1-Rad1 (9-1-1) complex and activation of downstream checkpoint kinases. Blocking either the NER or HR pathway abrogates checkpoint pathway activation and UV-C-induced apoptosis. Our results show that, following UV-C, multiple DNA repair pathways can cooperate to signal to the apoptotic machinery to eliminate potentially hazardous cells.