Nucleotide excision repair and the degradation of RNA pol II by the Caenorhabditis elegans XPA and Rsp5 orthologues, RAD-3 and WWP-1
Introduction
Spontaneous and environmental genotoxic stresses are responsible for inducing alterations in DNA, which can lead to genome instability. When DNA damage is detected in eukaryotic cells, cell division checkpoints are activated to provide time for lesions to be repaired; when DNA damage cannot be repaired, an apoptotic response may be elicited [1], [2]. Here, we focus on the nucleotide excision repair (NER) pathway, which is a versatile DNA damage response (DDR) pathway that removes a wide range of environmentally induced bulky DNA lesions, including cyclobutane pyrimidine dimers and (6–4) photoproducts.
NER is a multistep process involving at least 30 genes [3]. A subset of these genes is mutated in the rare human genetic disorders xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy. Patients affected by these disorders are usually hyper-photosensitive; however, they can also display a confounding spectrum of clinical features that are not always simply explained by differences in their underlying molecular defects [4], [5]. Mutations in any one of eight complementation groups, XP-A to G and variant, can cause XP [6]. Individuals with XP have an elevated susceptibility to develop skin cancer, and those with more severe forms of the disease show neurological degeneration [7]. By contrast, individuals affected with either CS or trichothiodystrophy have a normal cancer risk, although they display developmental and neurological problems [7]. CS is associated with defects in transcription-coupled repair (see below) [8], [9], involving the CSB and CSA proteins [3]. Trichothiodystrophy is associated with defects in the XPD and XPB helicase subunits of TFIIH [10], and also TFB5, the tenth subunit of TFIIH [11].
NER is composed of two sub-pathways based on the genomic location of the lesion: lesions on non-transcribed genomic DNA activate global genome repair, and lesions on the transcribed strand of expressed genes invoke transcription-coupled repair [3]. During global genome repair, the protein complexes UV-DDB and XPC/HR23B/Cen2 initiate repair and recruit the general transcription factor TFIIH and other repair proteins to the site of damage. Transcription-coupled repair is activated when RNA polymerase II (RNA pol II) complexes are stalled at UV lesions and attract CSB, which in turn, recruits TFIIH, XPA and other core NER factors and a complex including CSA. Prolonged stalling of RNA pol II in response to DNA damage [12], [13] or from elongation pausing [14], [15] leads to polyubiquitylation and degradation of the large RNA pol II subunit, Rpb1, by the proteasome [16], [17]. In Saccharomyces cerevisiae, polyubiquitylation and degradation of Rpb1 in response to UV damage is dependent on Def1, which encodes a novel protein that interacts with Rad26 (CSB homologue) [16], [17]. A human Def1 homologue has not been identified [16].
The enhanced cancer predisposition of XP patients can be readily explained by invoking genomic instability [18]. However, the basis for the neurodegenerative and developmental defects associated with XP are not completely understood, although it is apparent that some of the proteins involved, XPB, XPD and CSB, also play roles in basal transcription, which are distinct from their involvement in DDR [7]. Nonetheless, disruption of XPA, which so far is known to function only in NER, can also produce a full spectrum of XP symptoms [19]. This has led to the hypothesis that the neurodegenerative and developmental defects associated with the loss of NER function might be caused by the accumulation of cytotoxic NER-lesions, arising from oxidative stress in metabolically active and transcriptionally active tissues [20], [21].
In this paper, we have examined the role of NER during the development of the genetically tractable nematode Caenorhabditis elegans. C. elegans is a good model for such studies because homologues exist for many of the genes found in humans, which are involved in DDR, such as those involved in DNA repair and checkpoint responses [22]. Investigations with C. elegans also make it possible to examine how a defect in NER affects somatic cells in the absence of confounding contributions from apoptosis or cell cycle arrest [23], and across development in a whole organism. In C. elegans, an early genetic screen identified nine radiation sensitive (Rad) complementation groups [24], but to date only rad-5/clk-2 has been identified at the molecular level [25]. To learn more about genes that regulate the DDR to UV irradiation in C. elegans, we sought to determine the molecular identity of rad-3, which displays sensitivity to UV, but not to gamma irradiation [24]. We show for the first time that rad-3(mn157) is a null allele of the C. elegans orthologue of the XPA gene. In the absence of UV exposure, the development of xpa-1/rad-3 worms is virtually indistinguishable from wild type. By contrast to humans, we have observed no sign of ataxic neuronal defects in C. elegans xpa-1 mutants. Furthermore, xpa-1 mutants are not hypersensitive to oxidative stress and do not have a shortened lifespan. However, when xpa-1 animals are irradiated with UV they undergo an immediate growth arrest and display impaired survival and hypermutability. Surprisingly, growth arrested and transcriptionally quiescent dauer stage larvae survive after receiving UV doses that would be otherwise lethal to worms at other early stages of larval development. We further show that the UV-induced growth arrest and subsequent death of xpa-1 worms is correlated with transcriptional inhibition. This inhibition is associated with a decline in the level of the large RNA pol II subunit (AMA-1). We show that the degradation of AMA-1 is dependent on the activity of an E3 ubiquitin ligase encoded by the wwp-1 gene, and that UV sensitivity is exacerbated in the wwp-1 xpa-1 double mutant. These results extend our understanding of how defects in NER affect the development and perturb the DNA damage response of a whole organism at different life stages.
Section snippets
Nematode strains and handling
Strains were grown and maintained at 20 °C as described [26]. The following strains were used: wild type Bristol N2, RB1178: wwp-1(ok1102) I, SP482: rad-3(mn157) I, RB864: xpa-1(ok698) I, SP457: unc-93(e1500) III, SJ4005: zcIs4[hsp-4::GFP], TK22: mev-1(kn1) V, EG1285: lin-15(n765) oxIs12[unc-47::GFP + lin15(+)] X, VT765: maIs103[rnr::GFP + unc-36(+)], VT825 maIs113[cki-1::GFP + dpy-20(+)]. The wwp-1(ok1102) strain was backcrossed 3× and xpa-1(ok698) was backcrossed 5× prior to study.
Preparation of nucleic acids
C. elegans genomic
rad-3 is the C. elegans XPA homologue
To understand the mechanisms underlying the DDR to UV irradiation in the nematode C. elegans, we sought to determine the molecular identity of the rad-3 mutation. rad-3 is one of nine loci that was identified in a genetic screen for radiation sensitive animals [24]. We used a candidate gene approach to ask if it were possible to identify a gene within the genetic mapping interval determined for rad-3, which might be responsible for conferring a UV sensitivity phenotype. We found that the xpa-1
Discussion
The identification of the mutated gene responsible for causing the C. elegans rad-3 UV sensitivity phenotype has remained a mystery for over 25 years. Here, we have shown that rad-3 mutants are defective in NER because they carry a null allele of the xpa-1 gene. In humans, null alleles of XPA cause the most severe forms of XP [19]. Unlike other proteins involved in NER, XPA has no known role outside of DNA repair. Thus, the mutation in xpa-1 has allowed us to investigate how defects in NER
Conflict of interest
None.
Acknowledgements
We thank Phil Hartman for discussion. Some strains were provided by the Caenorhabditis Genetics Center (CGC), which is supported by the National Institute of Health National Center for Research Resources. J.A. was supported by a studentship from the University of Bristol and early support from the Sanger Institute (Hinxton). P.E.K. is supported by a Senior Non-Clinical Fellowship from the MRC.
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2021, Cell ReportsCitation Excerpt :These data exemplify the importance of TC-NER rather than GG-NER for maintaining transcriptional integrity and cell functionality in post-mitotic neurons, which likely correlates to the fact that neurodegeneration is a typical feature of human patients carrying mutations in TC-NER factors (Hoeijmakers, 2009; Karikkineth et al., 2017; Lans et al., 2019). Our results suggest that L1 larvae arrest development upon UV irradiation due to transcription arrest, which was previously reported to involve ERK1/2 mitogen-activated protein kinase (MAPK) signaling (Astin et al., 2008; Bianco and Schumacher, 2018; Lans and Vermeulen, 2011). Considering the importance of TC-NER in somatic cells, it is striking to note that loss of csb-1 in a wild-type or unc-119::xpf-1 background does not completely impair UV survival of L1 larvae (Figure 4F).
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