Xeroderma Pigmentosum

Xeroderma pigmentosum (XP) is defined by extreme sensitivity to sunlight, resulting in sunburn, pigment changes in the skin and a greatly elevated incidence of skin cancers. It is a rare autosomal recessive disorder and has been found in all continents and racial groups. Estimated incidences vary from 1 in 20, 000 in Japan to 1 in 250, 000 in the USA, and approximately 2.3 per million live births in Western Europe. The first features are either extreme sensitivity to sunlight, triggering severe sunburn, or, in patients who do not show this sun-sensitivity, abnormal lentiginosis (freckle-like pigmentation due to increased numbers of melanocytes) on sun-exposed areas. This is followed by areas of increased or decreased pigmentation, skin aging and multiple skin cancers, if the individuals are not protected from sunlight. A minority of patients show progressive neurological abnormalities. There are eight XP complementation groups, corresponding to eight genes, which, if defective, can result in XP. The products of these genes are involved in the repair of ultraviolet (UV)- induced damage in DNA. Seven of the gene products (XPA through G) are required to remove UV damage from the DNA. The eighth (XPV or DNA polymerase h) is required to replicate DNA containing unrepaired damage. There is wide variability in clinical features both between and within XP groups. Diagnosis is made clinically by the presence, from birth, of an acute and prolonged sunburn response at all exposed sites, unusually early lentiginosis in sun-exposed areas or onset of skin cancers at a young age. The clinical diagnosis is confirmed by cellular tests for defective DNA repair. These features distinguish XP from other photodermatoses such as solar urticaria and polymorphic light eruption, Cockayne Syndrome (no pigmentation changes, different repair defect) and other lentiginoses such as Peutz-Jeghers syndrome, Leopard syndrome and Carney complex (pigmentation not sunassociated), which are inherited in an autosomal dominant fashion. Antenatal diagnosis can be performed by measuring DNA repair or by mutation analysis in CVS cells or in amniocytes. Although there is no cure for XP, the skin effects can be minimised by rigorous protection from sunlight and early removal of pre-cancerous lesions. In the absence of neurological problems and with lifetime protection against sunlight, the prognosis is good. In patients with neurological problems, these are progressive, leading to disabilities and a shortened lifespan

A role for chromatin remodellers in replication of damaged DNA

In eukaryotic cells, replication past damaged sites in DNA is regulated by the ubiquitination of proliferating cell nuclear antigen (PCNA). Little is known about how this process is affected by chromatin structure. There are two isoforms of the Remodels the Structure of Chromatin (RSC) remodelling complex in yeast. We show that deletion of RSC2 results in a dramatic reduction in the level of PCNA ubiquitination after DNA-damaging treatments, whereas no such effect was observed after deletion of RSC1. Similarly, depletion of the BAF180 component of the corresponding PBAF (Polybromo BRG1 (Brahma-Related Gene 1) Associated Factor) complex in human cells led to a similar reduction in PCNA ubiquitination. Remarkably, we found that depletion of BAF180 resulted after UV-irradiation, in a reduction not only of ubiquitinated PCNA but also of chromatin-associated unmodified PCNA and Rad18 (the E3 ligase that ubiquitinates PCNA). This was accompanied by a modest decrease in fork progression. We propose a model to account for these findings that postulates an involvement of PBAF in repriming of replication downstream from replication forks blocked at sites of DNA damage. In support of this model, chromatin immunoprecipitation data show that the RSC complex in yeast is present in the vicinity of the replication forks, and by extrapolation, this is also likely to be the case for the PBAF complex in human cells

Cloning and characterisation of the rad9 DNA repair gene from Schizosaccharomyces pombe

The rad9.192 DNA repair mutant from the fission yeast, Schizosaccharomyces pombe, is sensitive to both UV and ionising radiation. The rad9 gene has been cloned by complementation of the gamma-ray sensitivity of the mutant cell line. A 4.3kb HindIII fragment was found to confer resistance to both types of radiation. The region of complementation was further localised to a 2.6kb HindIII-EcoRV fragment, which, by DNA sequence analysis, was found to contain sequences capable of coding for a 427 amino acid protein, if three introns were postulated to remove stop codons. The introns were confirmed by sequence analysis of cDNA clones and PCR products derived from cDNA. The product of transcription is a 1.6kb mRNA of low abundance. The putative rad9 protein shows no homology to any published sequence. A truncated protein is capable of complementing the radiation sensitivity of the rad9.192 mutant. Deletion of the gene is not lethal and the null allele has a similar phenotype to the rad9.192 mutant

Ubiquitination and deubiquitination of PCNA in response to stalling of the replication fork

Following exposure of human cells to DNA damaging agents that block the progress of the replication fork, mono-ubiquitination of PCNA mediates the switch from replicative DNA polymerases to polymerases specialised for translesion synthesis. We have shown that this modification of PCNA is necessary for the survival of cells after UV-irradiation and methyl methanesulfonate, that it is independent of cell cycle checkpoint activation, and that it persists after UV damage has been removed. In this Extra-view, we compare the regulation and biological significance of PCNA ubiquitination following treatments with UV light and the replication inhibitor hydroxyurea. We show that ubiquitination persists after removal of the replication block in both cases. With UV however, the persistence of ubiquitinated PCNA correlates with disappearance of the PCNA deubiquitinating enzyme USP1, whereas this is not the case for HU. Prevention of PCNA ubiquitination sensitises the cells to killing by both UV and HU

Nse2, a component of the Smc5-6 complex, is a SUMO ligase required for the response to DNA damage

The Schizosaccharomyces pombe SMC proteins Rad18 (Smc6) and Spr18 (Smc5) exist in a high-M(r) complex which also contains the non-SMC proteins Nse1, Nse2, Nse3, and Rad62. The Smc5-6 complex, which is essential for viability, is required for several aspects of DNA metabolism, including recombinational repair and maintenance of the DNA damage checkpoint. We have characterized Nse2 and show here that it is a SUMO ligase. Smc6 (Rad18) and Nse3, but not Smc5 (Spr18) or Nse1, are sumoylated in vitro in an Nse2-dependent manner, and Nse2 is itself autosumoylated, predominantly on the C-terminal part of the protein. Mutations of C195 and H197 in the Nse2 RING-finger-like motif abolish Nse2-dependent sumoylation. nse2.SA mutant cells, in which nse2.C195S-H197A is integrated as the sole copy of nse2, are viable, whereas the deletion of nse2 is lethal. Smc6 (Rad18) is sumoylated in vivo: the sumoylation level is increased upon exposure to DNA damage and is drastically reduced in the nse2.SA strain. Since nse2.SA cells are sensitive to DNA-damaging agents and to exposure to hydroxyurea, this implicates the Nse2-dependent sumoylation activity in DNA damage responses but not in the essential function of the Smc5-6 complex

A semi-automated non-radiactive system for measuring recovery of RNA synthesis and unscheduled DNA synthesis using ethynyluracil derivatives

Nucleotide excision repair (NER) removes the major UV-photolesions from cellular DNA. In humans, compromised NER activity is the cause of several photosensitive diseases, one of which is the skin-cancer predisposition disorder, xeroderma pigmentosum (XP). Two assays commonly used in measurement of NER activity are ‘unscheduled DNA synthesis (UDS)’, and ‘recovery of RNA synthesis (RRS)’, the latter being a specific measure of the transcription-coupled repair sub-pathway of NER. Both assays are key techniques for research in NER as well as in diagnoses of NER-related disorders. Until very recently, reliable methods for these assays involved measurements of incorporation of radio-labeled nucleosides. We have established non-radioactive procedures for determining UDS and RRS levels by incorporation of recently developed alkyne-conjugated nucleoside analogues, 5-ethynyl-2′-deoxyuridine (EdU) and 5-ethynyuridine (EU). EdU and EU are respectively used as alternatives for 3H-thymidine in UDS and for 3H-uridine in RRS. Based on these alkyne-nucleosides and an integrated image analyser, we have developed a semi-automated assay system for NER-activity. We demonstrate the utility of this system for NER-activity assessments of lymphoblastoid samples as well as primary fibroblasts. Potential use of the system for large-scale siRNA-screening for novel NER defects as well as for routine XP diagnosis are also considered

DNA repair, DNA replication and human disorders: A personal journey

I was born in 1946 and grew up in the industrial north-west of England close to the city of Manchester. My parents were German- Jewish refugees, who left Germany fairly early, in 1933. My father helped to establish and was one of the directors of a tannery, which made leather for shoes and handbags. This was part of a group of tanneries established first in Strasbourg by my great-grandfather Ferdinand Oppenheimer. I would describe my childhood and adolescent years as comfortable by general post-war standards. I went to a state primary school and obtained a scholarship to Manchester Grammar School (MGS), a fairly prestigious secondary school. As a child I was always interested in chemistry but had little interest in or knowledge of biology. The educational system in the UK at that time was such that one had to specialise very early and as a consequence I have had no formal biology education since the age of 12, something I have managed to hide reasonably successfully for the rest of my life! In my final two years at MGS I studied just physics, chemistry and mathematics and obtained a scholarship to Pembroke College, Cambridge (England) to study Natural Sciences, with the intention of becoming a chemist. In the second year at Cambridge, one of the options was a course on biochemistry. Having no real idea what this was, I read a book about it in the summer of 1965, and was truly astonished and excited to discover that the basis of life was just a bunch of rather complicated organic chemistry reactions. So I took the biochemistry course in my second year. By the end of that year, I was fed up with chemistry and for my final year I chose to do biochemistry rather than chemistry, a decision I have not regretted. The biochemistry lectures must have been pretty up-to-date, as we were told briefly about the discovery of DNA repair by Dick Setlow [1], a topic that seemed rather esoteric at the time

Impaired Translesion Synthesis in Xeroderma Pigmentosum Variant Extracts

Xeroderma pigmentosum variant (XPV) cells are characterized by a cellular defect in the ability to synthesize intact daughter DNA strands on damaged templates. Molecular mechanisms that facilitate replication fork progression on damaged DNA in normal cells are not well defined. In this study, we used single-stranded plasmid molecules containing a single N-2-acetylaminofluorene (AAF) adduct to analyze translesion synthesis (TLS) catalyzed by extracts of either normal or XPV primary skin fibroblasts. In one of the substrates, the single AAF adduct was located at the 3' end of a run of three guanines that was previously shown to induce deletion of one G by a slippage mechanism. Primer extension reactions performed by normal cellular extracts from four different individuals produced the same distinct pattern of TLS, with over 80% of the products resulting from the elongation of a slipped intermediate and the remaining 20% resulting from a nonslipped intermediate. In contrast, with cellular extracts from five different XPV patients, the TLS reaction was strongly reduced, yielding only low amounts of TLS via the nonslipped intermediate. With our second substrate, in which the AAF adduct was located at the first G in the run, thus preventing slippage from occurring, we confirmed that normal extracts were able to perform TLS 10-fold more efficiently than XPV extracts. These data demonstrate unequivocally that the defect in XPV cells resides in translesion synthesis independently of the slippage proces

Cloning and characterisation of the S.pombe rad15 gene, a homologue to the S.cerevisiae RAD3 and human ERCC2 genes

The RAD3 gene of Saccharomyces cerevisiae encodes an ATP-dependent 5' - 3' DNA helicase, which is involved in excision repair of ultraviolet radiation damage. By hybridisation of a Schizosaccharomyces pombe genomic library with a RAD3 gene probe we have isolated the S.pombe homologue of RAD3. We have also cloned the rad15 gene of S.pombe by complementation of radiation-sensitive phenotype of the rad15 mutant. Comparison of the restriction map and DNA sequence, shows that the S.pombe rad15 gene is identical to the gene homologous to S.cerevisiae RAD3, identified by hybridisation. The S.pombe rad15.P mutant is highly sensitive to UV radiation, but only slightly sensitive to ionising radiation, as expected for a mutant defective in excision repair. DNA sequence analysis of the rad15 gene indicates an open reading frame of 772 amino acids, and this is consistent with a transcript size of 2.6kb as detected by Northern analysis. The predicted rad15 protein has 65% identity to RAD3 and 55% identity to the human homologue ERCC2. This homology is particularly striking in the regions identified as being conserved in a group of DNA helicases. Gene deletion experiments indicate that, like the S.cerevisiae RAD3 gene, the S.pombe rad15 gene is essential for viability, suggesting that the protein product has a role in cell proliferation and not solely in DNA repair