Suppression of UV-induced apoptosis by the human DNA repair protein XPG.

The severe xeroderma pigmentosum/Cockayne syndrome (XP/CS) syndrome is caused by mutations in the XPB, XPD and XPG genes that encode the helicase subunits of TFIIH and the 3' endonuclease of nucleotide excision repair (NER). Because XPB and XPD have been implicated in p53-mediated apoptosis, we examined the possible involvement of XPG in this process. After ultraviolet light (UV) irradiation, primary fibroblasts of XP complementation group G (XP-G) individuals with CS enter apoptosis more readily than other NER-deficient cells, but this is unlinked to unrepaired damage. These XP-G/CS cells accumulate p53 post-UV but they fail to accumulate the 90/92 kDa isoforms of Mdm2 and their cellular distribution of Mdm2 is impaired. Apoptosis levels revert to wild type, Mdm2 90/92 kDa isoforms accumulate, and Mdm2 regains its normal post-UV nuclear location in transduced XP-G/CS cells expressing wild-type XPG, but not an XPG catalytic site mutant. These results suggest that XPG suppresses UV-induced apoptosis and that this suppression, most simply, requires its endonuclease function.

The XPG story.

I provide a personal account of the discovery, cloning and functional analyses of the human XPG gene. Mutations in this gene can give rise to the group G form of xeroderma pigmentosum (XP) and, in some cases, to severe early onset Cockayne syndrome (CS). The XPG protein has well established catalytic and structural roles in nucleotide excision repair (NER) and it acts as a cofactor for a DNA glycosylase that removes oxidised pyrimidines from DNA. XPG may also be involved in transcription-coupled repair of this kind of damage, in transcription by RNA polymerase II, and perhaps in other processes too. Our current knowledge of this important protein is largely based on some excellent, highly focussed science. But good luck, serendipity and scientific scandal have also made major contributions to this unfinished story.

Xeroderma pigmentosum group G with severe neurological involvement and features of Cockayne syndrome in infancy.

We describe a premature, small for gestational age infant girl with micropthalmia, bilateral congenital cataracts, hearing impairment, progressive somatic and neurodevelopmental arrest, and infantile spasms. She presented a massive photosensitive reaction with erythema and blistering after minimal sun exposure, which slowly gave place to small skin cancers. Her skin fibroblasts were 10-fold more sensitive than normal to UV exposure due to a severe deficiency in nucleotide excision repair. By complementation analysis, the patient XPCS4RO was assigned to the very rare xeroderma pigmentosum (XP) group G (XP-G). One allele of her XPG gene contained a 526C-->T transition that changed Gln-176 to a premature UAG stop codon. Only a minor fraction of XPG mRNA was encoded by this allele. The second, more significantly expressed XPG allele contained a 215C-->A transversion. This changed the highly conserved Pro-72 to a histidine, a substitution that would be expected to seriously impair the 3' endonuclease function of XPG in nucleotide excision repair. In cases suspected of having XP and/or early-onset Cockayne syndrome, extensive DNA repair studies should be performed to reach a correct diagnosis, thereby allowing reliable genetic counseling and prenatal diagnosis.

Conserved residues of human XPG protein important for nuclease activity and function in nucleotide excision repair.

The human XPG endonuclease cuts on the 3' side of a DNA lesion during nucleotide excision repair. Mutations in XPG can lead to the disorders xeroderma pigmentosum (XP) and Cockayne syndrome. XPG shares sequence similarities in two regions with a family of structure-specific nucleases and exonucleases. To begin defining its catalytic mechanism, we changed highly conserved residues and determined the effects on the endonuclease activity of isolated XPG, its function in open complex formation and dual incision reconstituted with purified proteins, and its ability to restore cellular resistance to UV light. The substitution A792V present in two XP complementation group G (XP-G) individuals reduced but did not abolish endonuclease activity, explaining their mild clinical phenotype. Isolated XPG proteins with Asp-77 or Glu-791 substitutions did not cleave DNA. In the reconstituted repair system, alanine substitutions at these positions permitted open complex formation but were inactive for 3' cleavage, whereas D77E and E791D proteins retained considerable activity. The function of each mutant protein in the reconstituted system was mirrored by its ability to restore UV resistance to XP-G cell lines. Hydrodynamic measurements indicated that XPG exists as a monomer in high salt conditions, but immunoprecipitation of intact and truncated XPG proteins showed that XPG polypeptides can interact with each other, suggesting dimerization as an element of XPG function. The mutation results define critical residues in the catalytic center of XPG and strongly suggest that key features of the strand cleavage mechanism and active site structure are shared by members of the nuclease family.

Base excision repair of oxidative DNA damage activated by XPG protein.

Oxidized pyrimidines in DNA are removed by a distinct base excision repair pathway initiated by the DNA glycosylase--AP lyase hNth1 in human cells. We have reconstituted this single-residue replacement pathway with recombinant proteins, including the AP endonuclease HAP1/APE, DNA polymerase beta, and DNA ligase III-XRCC1 heterodimer. With these proteins, the nucleotide excision repair enzyme XPG serves as a cofactor for the efficient function of hNth1. XPG protein promotes binding of hNth1 to damaged DNA. The stimulation of hNth1 activity is retained in XPG catalytic site mutants inactive in nucleotide excision repair. The data support the model that development of Cockayne syndrome in XP-G patients is related to inefficient excision of endogenous oxidative DNA damage.

Complementation of transformed fibroblasts from patients with combined xeroderma pigmentosum-Cockayne syndrome.

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are human hereditary disorders characterized at the cellular level by an inability to repair certain types of DNA damage. Usually, XP and CS are clinically and genetically distinct. However, in rare cases, CS patients have been shown to have mutations in genes that were previously linked to the development of XP. The linkage between XP and CS has been difficult to study because few permanent cell lines have been established from XP/CS patients. To generate permanent cell lines, primary fibroblast cultures from two patients, displaying characteristics associated with CS and belonging to XP complementation group G, were transformed with anorigin-of-replication-deficient simian virus 40 (SV40). The new cell lines, summation operatorXPCS1LVo- and summation operatorXPCS1ROo-,were characterized phenotypically and genotypically to verify that properties of the primary cells are preserved after transformation. The cell lines exhibited rapid growth in culture and were shown, by immunostaining, to express the SV40 T antigen. The summation operatorXPCS1LVo- and summation operatorXPCS1ROo- cell lines were hypersensitive to UV light and had an impaired ability to reactivate a UV-irradiated reporter gene. Using polymerase chain reaction (PCR) amplification and restriction enzyme cleavage, the summation operatorXPCS1ROo- cells were shown to retain the homozygous T deletion at XPG position 2972. This mutation also characterizes the parental primary cells and was evident in the XPG RNA. Finally, to characterize the XPG DNA repair deficiency in these cell lines, an episomal expression vector containing wild-type XPG cDNA was used to correct UV-induced damage in a beta-galactosidase reporter gene.

Efficient synthesis, termination and release of RNA polymerase III transcripts in Xenopus extracts depleted of La protein.

La proteins are conserved, abundant and predominantly nuclear phosphoproteins which bind to the 3'-U termini of newly synthesized RNA polymerase III transcripts. The human La protein has been implicated in the synthesis, termination and release of such transcripts. Here we examine the potential transcriptional properties of La in Xenopus laevis, using a homologous tRNA gene as template. Immunodepletion of La from cell-free extracts leads to the formation of tRNA precursors lacking 3'-U residues. This shortening can be uncoupled from RNA polymerase III transcription, indicating that it results from nuclease degradation rather than incomplete synthesis. Extracts containing <1% of the normal La protein content synthesize tRNA precursors just as well as complete extracts, with no change in termination efficiency, and the vast majority of these full-length transcripts are not associated with the template or with residual La protein. Hence, Xenopus La seems not to function as an initiation, termination or release factor for RNA polymerase III. Consistent with the recently discovered role of La in yeast tRNA maturation in vivo, recombinant Xenopus La prevents 3'-exonucleolytic degradation of tRNA precursors in vitro. A conserved RNA chaperone function may best explain the abundance of La in eukaryotic nuclei.

A common mutational pattern in Cockayne syndrome patients from xeroderma pigmentosum group G: implications for a second XPG function.

Xeroderma pigmentosum (XP) patients have defects in nucleotide excision repair (NER), the versatile repair pathway that removes UV-induced damage and other bulky DNA adducts. Patients with Cockayne syndrome (CS), another rare sun-sensitive disorder, are specifically defective in the preferential removal of damage from the transcribed strand of active genes, a process known as transcription-coupled repair. These two disorders are usually clinically and genetically distinct, but complementation analyses have assigned a few CS patients to the rare XP groups B, D, or G. The XPG gene encodes a structure-specific endonuclease that nicks damaged DNA 3' to the lesion during NER. Here we show that three XPG/CS patients had mutations that would produce severely truncated XPG proteins. In contrast, two sibling XPG patients without CS are able to make full-length XPG, but with a missense mutation that inactivates its function in NER. These results suggest that XPG/CS mutations abolish interactions required for a second important XPG function and that it is the loss of this second function that leads to the CS clinical phenotype.

Defective transcription-coupled repair of oxidative base damage in Cockayne syndrome patients from XP group G.

In normal human cells, damage due to ultraviolet light is preferentially removed from active genes by nucleotide excision repair (NER) in a transcription-coupled repair (TCR) process that requires the gene products defective in Cockayne syndrome (CS). Oxidative damage, including thymine glycols, is shown to be removed by TCR in cells from normal individuals and from xeroderma pigmentosum (XP)-A, XP-F, and XP-G patients who have NER defects but not from XP-G patients who have severe CS. Thus, TCR of oxidative damage requires an XPG function distinct from its NER endonuclease activity. These results raise the possibility that defective TCR of oxidative damage contributes to the developmental defects associated with CS.

A yeast RNA binding protein that resembles the human autoantigen La.

The La protein is a 47 kDa polypeptide that frequently acts as an autoantigen in systemic lupus erythematosus and Sjögren's syndrome patients. A key property of this protein is its association with the U-rich termini of newly synthesized RNA polymerase III transcripts. Here we characterize a 32 kDa protein from Saccharomyces cerevisiae that shows sequence similarity to the N termini of vertebrate La proteins. This yeast protein also functionally resembles La in that it binds preferentially in vitro to RNAs ending with a series of U residues, and at least 53 amino acids can be deleted from the C terminus without impeding this activity. Such RNA binding activity can be detected in crude yeast extracts by immunoprecipitation of ribonucleoprotein particles by an antibody to frog La protein. However, the same antibody fails to react with the 32 kDa protein. In addition, the gene encoding this protein is not essential for viability. Together, these results suggest that additional La homologue(s) exist in yeast.

Isolation of active recombinant XPG protein, a human DNA repair endonuclease.

Complementation group G of xeroderma pigmentosum (XP-G) is one of the most rare and phenotypically heterogeneous forms of this inherited disorder. XP-G patients vary from having a very mild defect in DNA repair to being severely affected, and a few cases are also associated with the neurological complications of Cockayne's syndrome. The XPG gene encodes an acidic protein with a predicted molecular mass of 133 kDa that confers normal UV resistance when expressed in XP-G cells. Here we report the isolation of full-length XPG as a soluble protein expressed from a recombinant baculovirus. The purified polypeptide corrects the DNA nucleotide excision repair defect of XP-G cell extracts in vitro, and it acts as a magnesium-dependent single-stranded DNA endonuclease. This is the first direct evidence for a human protein with properties that implicate it in the incision step of nucleotide excision repair.

Mutations that disable the DNA repair gene XPG in a xeroderma pigmentosum group G patient.

The human XPG (ERCC5) gene encodes a large acidic protein that corrects the ultraviolet light sensitivity of cells from both xeroderma pigmentosum complementation group G and rodent ERCC group 5. Here we characterize five XPG sequence alterations and a minor splicing defect in XP-G patient XP125LO. Three of these changes are polymorphic variants whereas the remaining two, one in each XPG allele, inactivate complementation in vivo. These single point mutations provide formal proof that defects in XPG give rise to the group G form of xeroderma pigmentosum, and their locations suggest ways in which this may occur.

La proteins from Xenopus laevis. cDNA cloning and developmental expression.

In mammalian nuclei, newly-synthesized RNA polymerase III transcripts are transiently associated with a phosphorylated polypeptide of approximately 50 kDa called the La protein. Here we provide evidence that the frog Xenopus laevis contains mRNAs for two highly related La proteins, each apparently encoded by a single gene. Both forms of the La protein contain the RNP-80 motif previously identified in many RNA binding proteins. The steady state levels of La mRNAs and protein are approximately constant in oocytes, eggs and embryos. This implies a progressive and severe decrease in these levels on a per cell basis during early development. In particular, neither the La mRNA nor protein level increases at the mid-blastula transition, the time when RNA polymerase III transcription first occurs during embryogenesis.

Complementation of the DNA repair defect in xeroderma pigmentosum group G cells by a human cDNA related to yeast RAD2.

Defects in human DNA repair proteins can give rise to the autosomal recessive disorders xeroderma pigmentosum (XP) and Cockayne's syndrome (CS), sometimes even together. Seven XP and three CS complementation groups have been identified that are thought to be due to mutations in genes from the nucleotide excision repair pathway. Here we isolate frog and human complementary DNAs that encode proteins resembling RAD2, a protein involved in this pathway in yeast. Alignment of these three polypeptides, together with two other RAD2 related proteins, reveals that their conserved sequences are largely confined to two regions. Expression of the human cDNA in vivo restores to normal the sensitivity to ultraviolet light and unscheduled DNA synthesis of lymphoblastoid cells from XP group G, but not CS group A. The XP-G correcting protein XPGC is generated from a messenger RNA of approximately 4 kilobases that is present in normal amounts in the XP-G cell line.

Oocyte and somatic tyrosine tRNA genes in Xenopus laevis.

Over a period of many months, Xenopus oocytes stockpile large quantities of tRNA for use during the first few hours of embryogenesis. To test the idea that these tRNAs are transcribed from one set of genes and that another set is used by somatic cells, we used synthetic oligonucleotides to analyze the sequence and steady-state levels of unspliced tyrosine tRNA precursors in Xenopus laevis oocytes, embryos, and cultured kidney cells. These analyses identify four kinds of tyrosine tRNA genes, two oocyte-type and two somatic-type, whose unspliced transcripts are distinguishable from one another by their different 5' leader and intervening sequences. The oocyte-type tyrosine tRNA precursors are present in oocytes, very abundant in gastrula embryos, but absent from postembryonic somatic cells. The somatic-type precursors are undetectable in oocytes but are found in gastrula and later stage embryos and in somatic cells. The major switch from oocyte-type to somatic-type transcripts occurs early during embryogenesis, between the midblastula transition and the onset of neurulation, but some oocyte-type precursors are also detectable in tadpoles.

Structure and transcription termination of a lysine tRNA gene from Xenopus laevis.

Termination of RNA polymerase III transcripts commonly occurs at clusters of T residues. A T4 tract located 72 base-pairs beyond a lysine tRNA gene from Xenopus laevis serves as an efficient termination site for the tRNA(Lys) precursors synthesized from this gene in homologous cell-free extracts. Nucleotides following this T tract influence the extent of read-through transcription in vitro, but in a way that differs from Xenopus 5 S RNA termination. Only approximately 50% of the transcripts initiated in vitro extend as far as this downstream T cluster. The remainder prematurely terminate at a second T4 tract located within the gene itself. The contrasting behaviour of these two T tracts in injected oocytes indicates that termination can be influenced by more than just RNA polymerase III alone, and that different components may contribute to, or hinder, termination at these sites. Prematurely terminated tRNA(Lys) transcripts are detectable in RNA from ovary tissue but not from a kidney cell line, suggesting that read-through transcription beyond intragenic T clusters can be modulated in vivo.