Stuart G. Clarkson

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.

Coordination of dual incision and repair synthesis in human nucleotide excision repair

Nucleotide excision repair (NER) requires the coordinated sequential assembly and actions of the involved proteins at sites of DNA damage. Following damage recognition, dual incision 5′ to the lesion by ERCC1‐XPF and 3′ to the lesion by XPG leads to the removal of a lesion‐containing oligonucleotide of about 30 nucleotides. The resulting single‐stranded DNA (ssDNA) gap on the undamaged strand is filled in by DNA repair synthesis. Here, we have asked how dual incision and repair synthesis are coordinated in human cells to avoid the exposure of potentially harmful ssDNA intermediates. Using catalytically inactive mutants of ERCC1‐XPF and XPG, we show that the 5′ incision by ERCC1‐XPF precedes the 3′ incision by XPG and that the initiation of repair synthesis does not require the catalytic activity of XPG. We propose that a defined order of dual incision and repair synthesis exists in human cells in the form of a ‘cut‐patch‐cut‐patch’ mechanism. This mechanism may aid the smooth progression through the NER pathway and contribute to genome integrity.

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.

Mutations of the yeast SUP4 tRNATyr Locus: Transcription of the mutant genes in vitro

Twenty-nine different SUP4-o tRNATyr genes with second-site mutations were transcribed in X. laevis cell-free RNA polymerase III transcription reactions, and the in vitro transcripts were analyzed by polyacrylamide gel electrophoresis. Nineteen mutant genes yield normal amounts of RNA that co-electrophorese with SUP4-o gene transcripts. RNA synthesized from a mutant gene lacing a single base pair migrated slightly faster in gels, as expected. The still shorter transcripts made from seven other mutant genes suggest that several mutations alter transcription starting or stopping points. Fingerprint analyses of transcripts from the two most extreme cases showed that premature termination occurred at new tracts of T residues resulting from the mutations. Two mutations significantly enhance transcription, and two mutations which alter the invariant C within the T psi CG sequence dramatically reduce SUP4-o gene transcription. The regions of the SUP4-o gene that surround these mutations are partially homologous to intragenic sequences in many other eucaryotic tRNA and 5S RNA genes. We hypothesize that these homologous sequences are recognized as promoter regions during RNA polymerase III transcription initiation.

Transcription initiation of eucaryotic transfer RNA Genes

RNA polymerase Ill (Pal Ill), an enzyme found in the nuclei of animals, plants and fungi, has been implicated in the in vivo transcription of 5s rRNA, pretRNA, some small viral RNAs and the cellular RNAs derived from certain middle-repetitive genomic sequences. It is a complex -700 kd protein composed of at least ten distinct subunits. The enzyme is not able to transcribe purified genes with fidelity by itself but requires additional components whose number, nature and modes of action are only just beginning to be characterized. Much more is known about the locations of the DNA signals that permit accurate transcription initiation of some Pol Ill genes. This information has come primarily from a two-step experimental approach. In the first step, DNA is progressively deleted from around and within the gene in question; the deleted sequences are then replaced by heterologous DNA. Alternatively, single-base changes or short deletions are introduced into the gene by in vivo or in vitro mutagenesis. The second step is then to assay the transcriptional effects of these sequence manipulations by microinjection of the mutant genes into the nuclei of frog oocytes or by their incubation in a variety of in vitro transcription systems. We shall compare and contrast the results of such analyses with several Pol Ill genes and discuss, in particular, the structure and function of the DNA signals important for tRNA gene transcription initiation. Pol III Promoters Are lntragenic Deletion analyses have shown that the only DNA sequences essential for transcription of a frog 5s RNA gene are located between residues 50 and 83 (Sakonju et al., Cell 79, 13-25, 1980; Bogenhagen et al., Cell 79, 27-35, 1980). A plausible basis for the mode of action of this intragenic promoter was provided by Engelke et al. (Cell 79, 717-728, 19801, who showed that the same region of the 5s RNA gene binds to a 5S-specific transcription factor. These results suggest that this 37 kd transcription factor, once bound, may interact with a Pol III molecule to position its catalytic sites on the transcription start point of the DNA. The same resection approach has shown that tRNA genes also contain intragenic promoters. Their maximum boundaries have been defined as residues 8 and 62 within the genes encoding the initiator tRNAMe’ (Hofstetter et al., Cell 24, 573-585, 1981) and tRNA% (Galli et al., Nature 294, 626-631, 1981) of X. laevis and the tRNAP”’ of Caenorhabditis elegans (Ciliberto et al., PNAS 79, 1195-l 199, 1982). Unlike their counterparts within 5s genes, however, the essential nucleotides are split into two sequence blocks that are set far apart. These sequences, termed the A and B blocks (Galli et al., op. cit.), have the approximate coordinates 8-19 and 52-62, respectively, by the standard system of numbering tRNA genes. Their locations with respect to the tRNA cloverleaf are indicated in Figure 1. Two lines of evidence in particular support the notion of discontinuous intragenic promoters: chimeric tRNA genes containing the 5’ half of one gene and the 3’ half of another can be transcribed well; and transcription can also occur after the replacement of the central region of tRNA genes with DNA of very different sequence (Ciliberto et al., op. cit.; Galli et al., op. cit.). These central regions do appear to have a spacing function, however, because the efficiency of transcription depends on the length of the replaced DNA, the optimal distance between the A and B blocks being 30-40 bp (Ciliberto et al., PNAS 79, 19211925, 1982). In natural tRNA genes this distance can vary from 31 to >74 bp, the variability being due to the length of the V arm and the presence within certain tRNA genes of an intervening sequence. Although not absolutely required for transcription (Wallace et al., Science 209, 1396-1400, 1980), intervening sequences may still influence the efficiency of this process by expanding the distance between the A and B blocks, thereby diminishing promoter strength. An unusual feature of tRNA gene promoters is thus the great latitude in distance between the A and B blocks. In contrast, the distance between the transcription start point and the A block is less variable (10-I 6 bp). Another intriguing feature of these sequences is their close correlation with the most con-

Definition of a Short Region of XPG Necessary for TFIIH Interaction and Stable Recruitment to Sites of UV Damage

ABSTRACT XPG is the human endonuclease that cuts 3′ to DNA lesions during nucleotide excision repair. Missense mutations in XPG can lead to xeroderma pigmentosum (XP), whereas truncated or unstable XPG proteins cause Cockayne syndrome (CS), normally yielding life spans of <7 years. One XP-G individual who had advanced XP/CS symptoms at 28 years has been identified. The genetic, biochemical, and cellular defects in this remarkable case provide insight into the onset of XP and CS, and they reveal a previously unrecognized property of XPG. Both of this individuals XPG alleles produce a severely truncated protein, but an infrequent alternative splice generates an XPG protein lacking seven internal amino acids, which can account for his very slight cellular UV resistance. Deletion of XPG amino acids 225 to 231 does not abolish structure-specific endonuclease activity. Instead, this region is essential for interaction with TFIIH and for the stable recruitment of XPG to sites of local UV damage after the prior recruitment of TFIIH. These results define a new functional domain of XPG, and they demonstrate that recruitment of DNA repair proteins to sites of damage does not necessarily lead to productive repair reactions. This observation has potential implications that extend beyond nucleotide excision repair.

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.

Chromosomal location of a major tRNA gene cluster of Xenopus laevis

In Xenopus laevis, genes encoding tRNAPhe, tRNATyr, tRNA1Met, tRNAAsn, tRNAAla, tRNALeu, and tRNALys are clustered within a 3.18-kb (kilobase) fragment of DNA. This fragment is tandemly repeated some 150 times in the haploid genome and its components are found outside the repeat only to a limited extent. The fragment hybridizes in situ to a single site very near the telomere on the long arm of one of the acrocentric chromosomes of the group comprising chromosomes 13–18. All the chromosomes of this group also hybridize with DNA coding for oocyte-specific 5S RNA. The tRNA gene cluster is slightly proximal to the cluster of 5S RNA genes.

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.

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.