Elsevier

DNA Repair

Volume 7, Issue 2, 1 February 2008, Pages 267-280
DNA Repair

Nucleotide excision repair and the degradation of RNA pol II by the Caenorhabditis elegans XPA and Rsp5 orthologues, RAD-3 and WWP-1

https://doi.org/10.1016/j.dnarep.2007.10.004Get rights and content

Abstract

The Caenorhabditis elegans rad-3 gene was identified in a genetic screen for radiation sensitive (rad) mutants. Here, we report that the UV sensitivity of rad-3 mutants is caused by a nonsense mutation in the C. elegans orthologue of the human nucleotide excision repair gene XPA. We have used the xpa-1/rad-3 mutant to examine how a defect in nucleotide excision repair (NER) perturbs development. We find that C. elegans carrying a mutation in xpa-1/rad-3 are hypersensitive and hypermutable in response to UV irradiation, but do not display hypersensitivity to oxidative stress or show obvious developmental abnormalities in the absence of UV exposure. Consistent with these observations, non-irradiated xpa-1 mutants have a similar lifespan as wild type. We further show that UV irradiated xpa-1 mutants undergo a stage-dependent decline in growth and survival, which is associated with a loss in transcriptional competence. Surprisingly, transcriptionally quiescent dauer stage larvae are able to survive a dose of UV irradiation, which is otherwise lethal to early stage larvae. We show that the loss of transcriptional competence in UV irradiated xpa-1 mutants is associated with the degradation of the large RNA polymerase II (RNA pol II) subunit, AMA-1, and have identified WWP-1 as the putative E3 ubiquitin ligase mediating this process. The absence of wwp-1 by itself does not cause sensitivity to UV irradiation, but it acts synergistically with a mutation in xpa-1 to enhance UV hypersensitivity.

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.

References (81)

  • L. Timmons et al.

    Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans

    Gene

    (2001)
  • X. Shen et al.

    Complementary signaling pathways regulate the unfolded protein response and are required for C. elegans development

    Cell

    (2001)
  • N. Miura et al.

    Identification and characterization of xpac protein, the gene product of the human XPAC (xeroderma pigmentosum group A complementing) gene

    J. Biol. Chem.

    (1991)
  • T. Shimamoto et al.

    Expression and functional analyses of the Dxpa gene, the Drosophila homolog of the human excision repair gene XPA

    J. Biol. Chem.

    (1995)
  • N. Ishii et al.

    A methyl viologen-sensitive mutant of the nematode Caenorhabditis elegans

    Mutat. Res.

    (1990)
  • T. Coohill et al.

    Ultraviolet mutagenesis of radiation-sensitive (rad) mutants of the nematode Caenorhabditis elegans

    Mutat. Res.

    (1988)
  • C. Kenyon

    The plasticity of aging: insights from long-lived mutants

    Cell

    (2005)
  • E.R. Hofmann et al.

    Caenorhabditis elegans HUS-1 Is a DNA damage checkpoint protein required for genome stability and EGL-1-mediated apoptosis

    Curr. Biol.

    (2002)
  • B. Schumacher et al.

    The C. elegans homolog of the p53 tumor suppressor is required for DNA damage-induced apoptosis

    Curr. Biol.

    (2001)
  • T. Sanford et al.

    RNA polymerase II from wild type and alpha-amanitin-resistant strains of Caenorhabditis elegans

    J. Biol. Chem.

    (1983)
  • K. Huang et al.

    A HECT domain ubiquitin ligase closely related to the mammalian protein WWP1 is essential for Caenorhabditis elegans embryogenesis

    Gene

    (2000)
  • A.R. Lehmann

    Replication of damaged DNA by translesion synthesis in human cells

    FEBS Lett.

    (2005)
  • P. Hartman et al.

    Does trans-lesion synthesis explain the UV-radiation resistance of DNA synthesis in C. elegans embryos?

    Mutat. Res.

    (1991)
  • T.J. Reape et al.

    Dauer larva recovery in the nematode Caenorhabditis elegans -I. The effect of mRNA synthesis inhibitors on recovery, growth and pharyngeal pumping

    Comp. Biochem. Physiol. B

    (1991)
  • Z. Luo et al.

    Ultraviolet radiation alters the phosphorylation of RNA polymerase II large subunit and accelerates its proteasome-dependent degradation

    Mutat. Res.

    (2001)
  • H. Murakami et al.

    DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts

    Biochem. J.

    (2000)
  • K.A. Nyberg et al.

    Toward maintaining the genome: DNA damage and replication checkpoints

    Annu. Rev. Genet.

    (2002)
  • E.C. Friedberg et al.

    DNA Repair and Mutagenesis

    (2005)
  • J.O. Andressoo et al.

    Nucleotide excision repair disorders and the balance between cancer and aging

    Cell Cycle

    (2006)
  • D. Bootsma et al.

    The Genetic Basis of Human Cancer

    (2002)
  • M. Berneburg et al.

    Xeroderma pigmentosum and related disorders: defects in DNA repair and transcription

    Adv. Genet.

    (2001)
  • A. van Hoffen et al.

    Deficient repair of the transcribed strand of active genes in Cockayne's syndrome cells

    Nucleic Acids Res.

    (1993)
  • G. Giglia-Mari et al.

    A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A

    Nat. Genet.

    (2004)
  • J.M. Huibregtse et al.

    The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase

    Proc. Natl. Acad. Sci. USA

    (1997)
  • S.L. Beaudenon et al.

    Rsp5 ubiquitin-protein ligase mediates DNA damage-induced degradation of the large subunit of RNA polymerase II in Saccharomyces cerevisiae

    Mol. Cell. Biol.

    (1999)
  • E.C. Woudstra et al.

    A Rad26-Def1 complex coordinates repair and RNA pol II proteolysis in response to DNA damage

    Nature

    (2002)
  • E.C. Friedberg

    How nucleotide excision repair protects against cancer

    Nat. Rev. Cancer

    (2001)
  • J.C. States et al.

    Distribution of mutations in the human xeroderma pigmentosum group A gene and their relationships to the functional regions of the DNA damage recognition protein

    Hum. Mutat.

    (1998)
  • M.S. Satoh et al.

    DNA excision-repair defect of xeroderma pigmentosum prevents removal of a class of oxygen free radical-induced base lesions

    Proc. Natl. Acad. Sci. USA

    (1993)
  • L. Stergiou et al.

    Death and more: DNA damage response pathways in the nematode C. elegans

    Cell Death Differ.

    (2004)
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