Publications by authors named "Alan R Lehmann"

80 Publications

Molecular analysis directs the prognosis, management and treatment of patients with xeroderma pigmentosum.

DNA Repair (Amst) 2020 09;93:102907

National Xeroderma Pigmentosum Service, Guy's and St Thomas' Foundation Trust, London SE1 9RT, UK.

Xeroderma pigmentosum (XP) is a well-studied disorder of (in most cases) nucleotide excision repair. The establishment in 2010 of a multidisciplinary XP clinic in the UK has enabled us to make a detailed analysis of genotype-phenotype relationships in XP patients and in several instances to make confident prognostic predictions. Splicing mutations in XPA and XPD and a specific amino acid change in XPD are associated with mild phenotypes, and individuals assigned to the XP-F group appear to have reduced pigmentation changes and a lower susceptibility to skin cancer than XPs in other groups. In an XP-C patient with advanced metastatic cancer arising from an angiosarcoma, molecular analysis of the tumour DNA suggested that immunotherapy, not normally recommended for angiosarcomas, might in this case be successful, and indeed the patient showed a dramatic recovery following immunotherapy treatment. These studies show that molecular analyses can improve the management, prognoses and therapy for individuals with XP.
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http://dx.doi.org/10.1016/j.dnarep.2020.102907DOI Listing
September 2020

UBR5 interacts with the replication fork and protects DNA replication from DNA polymerase η toxicity.

Nucleic Acids Res 2019 12;47(21):11268-11283

Istituto di Genetica Molecolare "Luigi Luca Cavalli-Sforza", CNR, Pavia, Italy.

Accurate DNA replication is critical for the maintenance of genome integrity and cellular survival. Cancer-associated alterations often involve key players of DNA replication and of the DNA damage-signalling cascade. Post-translational modifications play a fundamental role in coordinating replication and repair and central among them is ubiquitylation. We show that the E3 ligase UBR5 interacts with components of the replication fork, including the translesion synthesis (TLS) polymerase polη. Depletion of UBR5 leads to replication problems, such as slower S-phase progression, resulting in the accumulation of single stranded DNA. The effect of UBR5 knockdown is related to a mis-regulation in the pathway that controls the ubiquitylation of histone H2A (UbiH2A) and blocking this modification is sufficient to rescue the cells from replication problems. We show that the presence of polη is the main cause of replication defects and cell death when UBR5 is silenced. Finally, we unveil a novel interaction between polη and H2A suggesting that UbiH2A could be involved in polη recruitment to the chromatin and the regulation of TLS.
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http://dx.doi.org/10.1093/nar/gkz824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868395PMC
December 2019

Xeroderma pigmentosum: overview of pharmacology and novel therapeutic strategies for neurological symptoms.

Br J Pharmacol 2019 11 23;176(22):4293-4301. Epub 2019 Jan 23.

Ataxia Centre, Department of Clinical and Movement Neurosciences, University College London, Institute of Neurology London, London, UK.

Xeroderma pigmentosum (XP) encompasses a group of rare diseases characterized in most cases by malfunction of nucleotide excision repair (NER), which results in an increased sensitivity to UV radiation in affected individuals. Approximately 25-30% of XP patients present with neurological symptoms, such as sensorineural deafness, mental deterioration and ataxia. Although it is known that dysfunctional DNA repair is the primary pathogenesis in XP, growing evidence suggests that mitochondrial pathophysiology may also occur. This appears to be secondary to dysfunctional NER but may contribute to the neurodegenerative process in these patients. The available pharmacological treatments in XP mostly target the dermal manifestations of the disease. In the present review, we outline how current understanding of the pathophysiology of XP could be used to develop novel therapies to counteract the neurological symptoms. Moreover, the coexistence of cancer and neurodegeneration present in XP led us to focus on possible new avenues targeting mitochondrial pathophysiology. LINKED ARTICLES: This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc.
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http://dx.doi.org/10.1111/bph.14557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887903PMC
November 2019

Xeroderma pigmentosum is a definite cause of Huntington's disease-like syndrome.

Ann Clin Transl Neurol 2018 01 4;5(1):102-108. Epub 2017 Dec 4.

Ataxia Centre Department of Molecular Neuroscience University College London Institute of Neurology London WC1N 3BG United Kingdom.

Xeroderma pigmentosum is characterized by cutaneous, ophthalmological, and neurological features. Although it is typical of childhood, late presentations can mimic different neurodegenerative conditions. We report two families presenting as Huntington's disease-like syndromes. The first case (group G) presented with neuropsychiatric features, cognitive decline and chorea. Typical lentigines were only noticed after the neurological disease started. The second case (group B) presented adult-onset chorea and neuropsychiatric symptoms after an aggressive ocular melanoma. Xeroderma pigmentosum can manifest as a Huntington's Disease-like syndrome. Classic dermatological and oncological features have to be investigated in choreic patients with negative genetic tests for Huntington's disease-like phenotypes.
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http://dx.doi.org/10.1002/acn3.511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771320PMC
January 2018

Phosphorylation regulates human polη stability and damage bypass throughout the cell cycle.

Nucleic Acids Res 2017 Sep;45(16):9441-9454

Istituto di Genetica Molecolare-CNR, 27100, Pavia, Italy.

DNA translesion synthesis (TLS) is a crucial damage tolerance pathway that oversees the completion of DNA replication in the presence of DNA damage. TLS polymerases are capable of bypassing a distorted template but they are generally considered inaccurate and they need to be tightly regulated. We have previously shown that polη is phosphorylated on Serine 601 after DNA damage and we have demonstrated that this modification is important for efficient damage bypass. Here we report that polη is also phosphorylated by CDK2, in the absence of damage, in a cell cycle-dependent manner and we identify serine 687 as an important residue targeted by the kinase. We discover that phosphorylation on serine 687 regulates the stability of the polymerase during the cell cycle, allowing it to accumulate in late S and G2 when productive TLS is critical for cell survival. Furthermore, we show that alongside the phosphorylation of S601, the phosphorylation of S687 and S510, S512 and/or S514 are important for damage bypass and cell survival after UV irradiation. Taken together our results provide new insights into how cells can, at different times, modulate DNA TLS for improved cell survival.
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http://dx.doi.org/10.1093/nar/gkx619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766190PMC
September 2017

Specialized interfaces of Smc5/6 control hinge stability and DNA association.

Nat Commun 2017 01 30;8:14011. Epub 2017 Jan 30.

Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, BN1 9RQ, UK.

The Structural Maintenance of Chromosomes (SMC) complexes: cohesin, condensin and Smc5/6 are involved in the organization of higher-order chromosome structure-which is essential for accurate chromosome duplication and segregation. Each complex is scaffolded by a specific SMC protein dimer (heterodimer in eukaryotes) held together via their hinge domains. Here we show that the Smc5/6-hinge, like those of cohesin and condensin, also forms a toroidal structure but with distinctive subunit interfaces absent from the other SMC complexes; an unusual 'molecular latch' and a functional 'hub'. Defined mutations in these interfaces cause severe phenotypic effects with sensitivity to DNA-damaging agents in fission yeast and reduced viability in human cells. We show that the Smc5/6-hinge complex binds preferentially to ssDNA and that this interaction is affected by both 'latch' and 'hub' mutations, suggesting a key role for these unique features in controlling DNA association by the Smc5/6 complex.
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http://dx.doi.org/10.1038/ncomms14011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5290277PMC
January 2017

Destabilized SMC5/6 complex leads to chromosome breakage syndrome with severe lung disease.

J Clin Invest 2016 08 18;126(8):2881-92. Epub 2016 Jul 18.

The structural maintenance of chromosomes (SMC) family of proteins supports mitotic proliferation, meiosis, and DNA repair to control genomic stability. Impairments in chromosome maintenance are linked to rare chromosome breakage disorders. Here, we have identified a chromosome breakage syndrome associated with severe lung disease in early childhood. Four children from two unrelated kindreds died of severe pulmonary disease during infancy following viral pneumonia with evidence of combined T and B cell immunodeficiency. Whole exome sequencing revealed biallelic missense mutations in the NSMCE3 (also known as NDNL2) gene, which encodes a subunit of the SMC5/6 complex that is essential for DNA damage response and chromosome segregation. The NSMCE3 mutations disrupted interactions within the SMC5/6 complex, leading to destabilization of the complex. Patient cells showed chromosome rearrangements, micronuclei, sensitivity to replication stress and DNA damage, and defective homologous recombination. This work associates missense mutations in NSMCE3 with an autosomal recessive chromosome breakage syndrome that leads to defective T and B cell function and acute respiratory distress syndrome in early childhood.
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http://dx.doi.org/10.1172/JCI82890DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4966312PMC
August 2016

GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy.

Am J Hum Genet 2016 Apr 17;98(4):627-42. Epub 2016 Mar 17.

Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Via Abbiategrasso 207, 27100 Pavia, Italy. Electronic address:

The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.
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http://dx.doi.org/10.1016/j.ajhg.2016.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833217PMC
April 2016

Deep phenotyping of 89 xeroderma pigmentosum patients reveals unexpected heterogeneity dependent on the precise molecular defect.

Proc Natl Acad Sci U S A 2016 Mar 16;113(9):E1236-45. Epub 2016 Feb 16.

Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, United Kingdom;

Xeroderma pigmentosum (XP) is a rare DNA repair disorder characterized by increased susceptibility to UV radiation (UVR)-induced skin pigmentation, skin cancers, ocular surface disease, and, in some patients, sunburn and neurological degeneration. Genetically, it is assigned to eight complementation groups (XP-A to -G and variant). For the last 5 y, the UK national multidisciplinary XP service has provided follow-up for 89 XP patients, representing most of the XP patients in the United Kingdom. Causative mutations, DNA repair levels, and more than 60 clinical variables relating to dermatology, ophthalmology, and neurology have been measured, using scoring systems to categorize disease severity. This deep phenotyping has revealed unanticipated heterogeneity of clinical features, between and within complementation groups. Skin cancer is most common in XP-C, XP-E, and XP-V patients, previously considered to be the milder groups based on cellular analyses. These patients have normal sunburn reactions and are therefore diagnosed later and are less likely to adhere to UVR protection. XP-C patients are specifically hypersensitive to ocular damage, and XP-F and XP-G patients appear to be much less susceptible to skin cancer than other XP groups. Within XP groups, different mutations confer susceptibility or resistance to neurological damage. Our findings on this large cohort of XP patients under long-term follow-up reveal that XP is more heterogeneous than has previously been appreciated. Our data now enable provision of personalized prognostic information and management advice for each XP patient, as well as providing new insights into the functions of the XP proteins.
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http://dx.doi.org/10.1073/pnas.1519444113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780618PMC
March 2016

A Distinct Genotype of XP Complementation Group A: Surprisingly Mild Phenotype Highly Prevalent in Northern India/Pakistan/Afghanistan.

J Invest Dermatol 2016 Apr 29;136(4):869-872. Epub 2015 Dec 29.

National Xeroderma Pigmentosum Service, Department of Photodermatology, St. John's Institute of Dermatology, Guy's and St. Thomas' NHS Foundation Trust, London, United Kingdom. Electronic address:

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http://dx.doi.org/10.1016/j.jid.2015.12.031DOI Listing
April 2016

Chromatin association of the SMC5/6 complex is dependent on binding of its NSE3 subunit to DNA.

Nucleic Acids Res 2016 Feb 7;44(3):1064-79. Epub 2015 Oct 7.

Mendel Centre for Plant Genomics and Proteomics, Central European Institute of Technology, Masaryk University, Kamenice 5, Brno, 62500, Czech Republic Laboratory of Functional Genomics and Proteomics, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kotlarska 2, Brno, 61137, Czech Republic

SMC5/6 is a highly conserved protein complex related to cohesin and condensin, which are the key components of higher-order chromatin structures. The SMC5/6 complex is essential for proliferation in yeast and is involved in replication fork stability and processing. However, the precise mechanism of action of SMC5/6 is not known. Here we present evidence that the NSE1/NSE3/NSE4 sub-complex of SMC5/6 binds to double-stranded DNA without any preference for DNA-replication/recombination intermediates. Mutations of key basic residues within the NSE1/NSE3/NSE4 DNA-binding surface reduce binding to DNA in vitro. Their introduction into the Schizosaccharomyces pombe genome results in cell death or hypersensitivity to DNA damaging agents. Chromatin immunoprecipitation analysis of the hypomorphic nse3 DNA-binding mutant shows a reduced association of fission yeast SMC5/6 with chromatin. Based on our results, we propose a model for loading of the SMC5/6 complex onto the chromatin.
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http://dx.doi.org/10.1093/nar/gkv1021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756808PMC
February 2016

XRCC4 deficiency in human subjects causes a marked neurological phenotype but no overt immunodeficiency.

J Allergy Clin Immunol 2015 Oct 5;136(4):1007-17. Epub 2015 Aug 5.

Department of Genetics, Research Institute of Environmental Medicine (RIeM), Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan; Nagasaki University Research Centre for Genomic Instability and Carcinogenesis (NRGIC), Nagasaki, Japan; Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University, Nagasaki, Japan; Microbial Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, Mishima, Japan. Electronic address:

Background: Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair mechanism in human cells. The final rejoining step requires DNA ligase IV (LIG4) together with the partner proteins X-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor. Patients with mutations in genes encoding LIG4, XRCC4-like factor, or the other NHEJ proteins DNA-dependent protein kinase catalytic subunit and Artemis are DSB repair defective and immunodeficient because of the requirement for NHEJ during V(D)J recombination.

Objective: We found a patient displaying microcephaly and progressive ataxia but a normal immune response. We sought to determine pathogenic mutations and to describe the molecular pathogenesis of the patient.

Methods: We performed next-generation exome sequencing. We evaluated the DSB repair activities and V(D)J recombination capacity of the patient's cells, as well as performing a standard blood immunologic characterization.

Results: We identified causal mutations in the XRCC4 gene. The patient's cells are radiosensitive and display the most severe DSB repair defect we have encountered using patient-derived cell lines. In marked contrast, a V(D)J recombination plasmid assay revealed that the patient's cells did not display the junction abnormalities that are characteristic of other NHEJ-defective cell lines. The mutant protein can interact efficiently with LIG4 and functions normally in in vitro assays and when transiently expressed in vivo. However, the mutation makes the protein unstable, and it undergoes proteasome-mediated degradation.

Conclusion: Our findings reveal a novel separation of impact phenotype: there is a pronounced DSB repair defect and marked clinical neurological manifestation but no clinical immunodeficiency.
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http://dx.doi.org/10.1016/j.jaci.2015.06.007DOI Listing
October 2015

TFIIH-dependent MMP-1 overexpression in trichothiodystrophy leads to extracellular matrix alterations in patient skin.

Proc Natl Acad Sci U S A 2015 Feb 20;112(5):1499-504. Epub 2015 Jan 20.

Istituto di Genetica Molecolare Consiglio Nazionale delle Ricerche, Pavia 27100, Italy;

Mutations in the XPD subunit of the DNA repair/transcription factor TFIIH result in distinct clinical entities, including the cancer-prone xeroderma pigmentosum (XP) and the multisystem disorder trichothiodystrophy (TTD), which share only cutaneous photosensitivity. Gene-expression profiles of primary dermal fibroblasts revealed overexpression of matrix metalloproteinase 1 (MMP-1), the gene encoding the metalloproteinase that degrades the interstitial collagens of the extracellular matrix (ECM), in TTD patients mutated in XPD compared with their healthy parents. The defect is observed in TTD and not in XP and is specific for fibroblasts, which are the main producers of dermal ECM. MMP-1 transcriptional up-regulation in TTD is caused by an erroneous signaling mediated by retinoic acid receptors on the MMP-1 promoter and leads to hypersecretion of active MMP-1 enzyme and degradation of collagen type I in the ECM of cell/tissue systems and TTD patient skin. In agreement with the well-known role of ECM in eliciting signaling events controlling cell behavior and tissue homeostasis, ECM alterations in TTD were shown to impact on the migration and wound-healing properties of patient dermal fibroblasts. The presence of a specific inhibitor of MMP activity was sufficient to restore normal cell migration, thus providing a potential approach for therapeutic strategies. This study highlights the relevance of ECM anomalies in TTD pathogenesis and in the phenotypic differences between TTD and XP.
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http://dx.doi.org/10.1073/pnas.1416181112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4321311PMC
February 2015

The melanoma-associated antigen 1 (MAGEA1) protein stimulates the E3 ubiquitin-ligase activity of TRIM31 within a TRIM31-MAGEA1-NSE4 complex.

Cell Cycle 2015 ;14(6):920-30

a From the Mendel Center for Plant Genomics and Proteomics; Central European Institute of Technology; Masaryk University ; Brno , Czech Republic.

The MAGE (Melanoma-associated antigen) protein family members are structurally related to each other by a MAGE-homology domain comprised of 2 winged helix motifs WH/A and WH/B. This family specifically evolved in placental mammals although single homologs designated NSE3 (non-SMC element) exist in most eukaryotes. NSE3, together with its partner proteins NSE1 and NSE4 form a tight subcomplex of the structural maintenance of chromosomes SMC5-6 complex. Previously, we showed that interactions of the WH/B motif of the MAGE proteins with their NSE4/EID partners are evolutionarily conserved (including the MAGEA1-NSE4 interaction). In contrast, the interaction of the WH/A motif of NSE3 with NSE1 diverged in the MAGE paralogs. We hypothesized that the MAGE paralogs acquired new RING-finger-containing partners through their evolution and form MAGE complexes reminiscent of NSE1-NSE3-NSE4 trimers. In this work, we employed the yeast 2-hybrid system to screen a human RING-finger protein library against several MAGE baits. We identified a number of potential MAGE-RING interactions and confirmed several of them (MDM4, PCGF6, RNF166, TRAF6, TRIM8, TRIM31, TRIM41) in co-immunoprecipitation experiments. Among these MAGE-RING pairs, we chose to examine MAGEA1-TRIM31 in detail and showed that both WH/A and WH/B motifs of MAGEA1 bind to the coiled-coil domain of TRIM31 and that MAGEA1 interaction stimulates TRIM31 ubiquitin-ligase activity. In addition, TRIM31 directly binds to NSE4, suggesting the existence of a TRIM31-MAGEA1-NSE4 complex reminiscent of the NSE1-NSE3-NSE4 trimer. These results suggest that MAGEA1 functions as a co-factor of TRIM31 ubiquitin-ligase and that the TRIM31-MAGEA1-NSE4 complex may have evolved from an ancestral NSE1-NSE3-NSE4 complex.
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http://dx.doi.org/10.1080/15384101.2014.1000112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4614679PMC
December 2015

Transcription restores DNA repair to heterochromatin, determining regional mutation rates in cancer genomes.

Cell Rep 2014 Nov 20;9(4):1228-34. Epub 2014 Nov 20.

Department of Dermatology, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Somatic mutations in cancer are more frequent in heterochromatic and late-replicating regions of the genome. We report that regional disparities in mutation density are virtually abolished within transcriptionally silent genomic regions of cutaneous squamous cell carcinomas (cSCCs) arising in an XPC(-/-) background. XPC(-/-) cells lack global genome nucleotide excision repair (GG-NER), thus establishing differential access of DNA repair machinery within chromatin-rich regions of the genome as the primary cause for the regional disparity. Strikingly, we find that increasing levels of transcription reduce mutation prevalence on both strands of gene bodies embedded within H3K9me3-dense regions, and only to those levels observed in H3K9me3-sparse regions, also in an XPC-dependent manner. Therefore, transcription appears to reduce mutation prevalence specifically by relieving the constraints imposed by chromatin structure on DNA repair. We model this relationship among transcription, chromatin state, and DNA repair, revealing a new, personalized determinant of cancer risk.
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http://dx.doi.org/10.1016/j.celrep.2014.10.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4254608PMC
November 2014

Xeroderma pigmentosum in the United kingdom.

Authors:
Alan R Lehmann

Photochem Photobiol 2015 Mar-Apr;91(2):484-5. Epub 2014 Jul 14.

Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, BN1 9RQ, UK.

The seminal discovery by James Cleaver of defective DNA repair in xeroderma pigmentosum (XP) opened up an ever-expanding field of DNA repair-related disorders. In addition, it put XP on the map and has led to improved diagnosis, care and management of affected patients. In the United Kingdom, we recently established a multidisciplinary specialist clinic for XP patients. All XP patients in the United Kingdom are able to visit the clinic where they are examined and advised by a team of specialists with detailed knowledge of the different aspects of XP.
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http://dx.doi.org/10.1111/php.12301DOI Listing
September 2015

Hypomorphic PCNA mutation underlies a human DNA repair disorder.

J Clin Invest 2014 Jul 9;124(7):3137-46. Epub 2014 Jun 9.

Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA's interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration.
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http://dx.doi.org/10.1172/JCI74593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4071375PMC
July 2014

Malfunction of nuclease ERCC1-XPF results in diverse clinical manifestations and causes Cockayne syndrome, xeroderma pigmentosum, and Fanconi anemia.

Am J Hum Genet 2013 May 25;92(5):807-19. Epub 2013 Apr 25.

Department of Plastic and Reconstructive Surgery, Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan.

Cockayne syndrome (CS) is a genetic disorder characterized by developmental abnormalities and photodermatosis resulting from the lack of transcription-coupled nucleotide excision repair, which is responsible for the removal of photodamage from actively transcribed genes. To date, all identified causative mutations for CS have been in the two known CS-associated genes, ERCC8 (CSA) and ERCC6 (CSB). For the rare combined xeroderma pigmentosum (XP) and CS phenotype, all identified mutations are in three of the XP-associated genes, ERCC3 (XPB), ERCC2 (XPD), and ERCC5 (XPG). In a previous report, we identified several CS cases who did not have mutations in any of these genes. In this paper, we describe three CS individuals deficient in ERCC1 or ERCC4 (XPF). Remarkably, one of these individuals with XP complementation group F (XP-F) had clinical features of three different DNA-repair disorders--CS, XP, and Fanconi anemia (FA). Our results, together with those from Bogliolo et al., who describe XPF alterations resulting in FA alone, indicate a multifunctional role for XPF.
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http://dx.doi.org/10.1016/j.ajhg.2013.04.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3644632PMC
May 2013

SMC6 is an essential gene in mice, but a hypomorphic mutant in the ATPase domain has a mild phenotype with a range of subtle abnormalities.

DNA Repair (Amst) 2013 May 18;12(5):356-66. Epub 2013 Mar 18.

Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, UK.

Smc5-6 is a highly conserved protein complex related to cohesin and condensin involved in the structural maintenance of chromosomes. In yeasts the Smc5-6 complex is essential for proliferation and is involved in DNA repair and homologous recombination. siRNA depletion of genes involved in the Smc5-6 complex in cultured mammalian cells results in sensitivity to some DNA damaging agents. In order to gain further insight into its role in mammals we have generated mice mutated in the Smc6 gene. A complete knockout resulted in early embryonic lethality, demonstrating that this gene is essential in mammals. However, mutation of the highly conserved serine-994 to alanine in the ATP hydrolysis motif in the SMC6 C-terminal domain, resulted in mice with a surprisingly mild phenotype. With the neo gene selection marker in the intron following the mutation, resulting in reduced expression of the SMC6 gene, the mice were reduced in size, but fertile and had normal lifespans. When the neo gene was removed, the mice had normal size, but detailed phenotypic analysis revealed minor abnormalities in glucose tolerance, haematopoiesis, nociception and global gene expression patterns. Embryonic fibroblasts derived from the ser994 mutant mice were not sensitive to killing by a range of DNA damaging agents, but they were sensitive to the induction of sister chromatid exchanges induced by ultraviolet light or mitomycin C. They also accumulated more oxidative damage than wild-type cells.
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http://dx.doi.org/10.1016/j.dnarep.2013.02.006DOI Listing
May 2013

A role for chromatin remodellers in replication of damaged DNA.

Nucleic Acids Res 2012 Aug 25;40(15):7393-403. Epub 2012 May 25.

Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, UK.

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.
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http://dx.doi.org/10.1093/nar/gks453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3424576PMC
August 2012

DNA repair, DNA replication and human disorders: a personal journey.

Authors:
Alan R Lehmann

DNA Repair (Amst) 2012 Apr;11(4):328-34

Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, UK.

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http://dx.doi.org/10.1016/j.dnarep.2011.05.008DOI Listing
April 2012

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair.

Nat Genet 2012 May;44(5):586-92

Nagasaki University Research Centre for Genomic Instability and Carcinogenesis, Nagasaki, Japan.

UV-sensitive syndrome (UV(S)S) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma. Despite mild clinical features, cells from individuals with UV(S)S, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER), which removes DNA damage in actively transcribed genes. Three of the seven known UV(S)S cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively). The remaining four individuals with UVSS , one of whom is described for the first time here, formed a separate UV(S)S-A complementation group; however, the responsible gene was unknown. Using exome sequencing, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UV(S)S-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER–deficient disorders.
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http://dx.doi.org/10.1038/ng.2229DOI Listing
May 2012

Y-family DNA polymerases and their role in tolerance of cellular DNA damage.

Nat Rev Mol Cell Biol 2012 Feb 23;13(3):141-52. Epub 2012 Feb 23.

Division of Protein and Nucleic Acid Chemistry, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.

The past 15 years have seen an explosion in our understanding of how cells replicate damaged DNA and how this can lead to mutagenesis. The Y-family DNA polymerases lie at the heart of this process, which is commonly known as translesion synthesis. This family of polymerases has unique features that enable them to synthesize DNA past damaged bases. However, as they exhibit low fidelity when copying undamaged DNA, it is essential that they are only called into play when they are absolutely required. Several layers of regulation ensure that this is achieved.
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http://dx.doi.org/10.1038/nrm3289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3630503PMC
February 2012

Xeroderma pigmentosum.

Orphanet J Rare Dis 2011 Nov 1;6:70. Epub 2011 Nov 1.

Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, UK.

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 η) 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 sun-associated), 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.
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http://dx.doi.org/10.1186/1750-1172-6-70DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3221642PMC
November 2011

Human exonuclease 1 connects nucleotide excision repair (NER) processing with checkpoint activation in response to UV irradiation.

Proc Natl Acad Sci U S A 2011 Aug 1;108(33):13647-52. Epub 2011 Aug 1.

Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, 20133 Milano, Italy.

UV light induces DNA lesions, which are removed by nucleotide excision repair (NER). Exonuclease 1 (EXO1) is highly conserved from yeast to human and is implicated in numerous DNA metabolic pathways, including repair, recombination, replication, and telomere maintenance. Here we show that hEXO1 is involved in the cellular response to UV irradiation in human cells. After local UV irradiation, fluorescent-tagged hEXO1 localizes, together with NER factors, at the sites of damage in nonreplicating cells. hEXO1 accumulation requires XPF-dependent processing of UV-induced lesions and is enhanced by inhibition of DNA repair synthesis. In nonreplicating cells, depletion of hEXO1 reduces unscheduled DNA synthesis after UV irradiation, prevents ubiquitylation of histone H2A, and impairs activation of the checkpoint signal transduction cascade in response to UV damage. These findings reveal a key role for hEXO1 in the UV-induced DNA damage response linking NER to checkpoint activation in human cells.
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http://dx.doi.org/10.1073/pnas.1108547108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158162PMC
August 2011

Ubiquitin-family modifications in the replication of DNA damage.

Authors:
Alan R Lehmann

FEBS Lett 2011 Sep 23;585(18):2772-9. Epub 2011 Jun 23.

Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, UK.

The cell uses specialised Y-family DNA polymerases or damage avoidance mechanisms to replicate past damaged sites in DNA. These processes are under complex regulatory systems, which employ different types of post-translational modification. All the Y-family polymerases have ubiquitin binding domains that bind to mono-ubiquitinated PCNA to effect the switching from replicative to Y-family polymerase. Ubiquitination and de-ubiquitination of PCNA are tightly regulated. There is also evidence for another as yet unidentified ubiquitinated protein being involved in recruitment of Y-family polymerases to chromatin. Poly-ubiquitination of PCNA stimulates damage avoidance, and, at least in yeast, PCNA is SUMOylated to prevent unwanted recombination events at the replication fork. The Y-family polymerases themselves can be ubiquitinated and, in the case of DNA polymerase η, this results in the polymerase being excluded from chromatin.
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http://dx.doi.org/10.1016/j.febslet.2011.06.005DOI Listing
September 2011

DNA polymerases and repair synthesis in NER in human cells.

Authors:
Alan R Lehmann

DNA Repair (Amst) 2011 Jul 20;10(7):730-3. Epub 2011 May 20.

Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, UK.

The late steps of nucleotide excision repair, following incisions to remove the damaged section of DNA, comprise repair synthesis and ligation. In vitro and in vivo studies have shown the size of the repaired patch to be about 30 nucleotides. In vitro studies implicated the replicative polymerases in repair synthesis, but recent in vivo data have shown that several DNA polymerases and ligases are involved in these steps in human cells.
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http://dx.doi.org/10.1016/j.dnarep.2011.04.023DOI Listing
July 2011

Interactions between the Nse3 and Nse4 components of the SMC5-6 complex identify evolutionarily conserved interactions between MAGE and EID Families.

PLoS One 2011 Feb 25;6(2):e17270. Epub 2011 Feb 25.

Genome Damage and Stability Centre, University of Sussex, Brighton, United Kingdom.

Background: The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6-8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors.

Methodology/principal Findings: Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins.

Conclusions/significance: We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0017270PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3045436PMC
February 2011

ATR-mediated phosphorylation of DNA polymerase η is needed for efficient recovery from UV damage.

J Cell Biol 2011 Jan 17;192(2):219-27. Epub 2011 Jan 17.

Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ, England, UK.

DNA polymerase η (polη) belongs to the Y-family of DNA polymerases and facilitates translesion synthesis past UV damage. We show that, after UV irradiation, polη becomes phosphorylated at Ser601 by the ataxia-telangiectasia mutated and Rad3-related (ATR) kinase. DNA damage-induced phosphorylation of polη depends on its physical interaction with Rad18 but is independent of PCNA monoubiquitination. It requires the ubiquitin-binding domain of polη but not its PCNA-interacting motif. ATR-dependent phosphorylation of polη is necessary to restore normal survival and postreplication repair after ultraviolet irradiation in xeroderma pigmentosum variant fibroblasts, and is involved in the checkpoint response to UV damage. Taken together, our results provide evidence for a link between DNA damage-induced checkpoint activation and translesion synthesis in mammalian cells.
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http://dx.doi.org/10.1083/jcb.201008076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172178PMC
January 2011

Structure and mechanism of human DNA polymerase eta.

Nature 2010 Jun;465(7301):1044-8

Laboratory of Molecular Biology, NIDDK, NIH, 9000 Rockville Pike, Building 5, Room B103, Bethesda, Maryland 20892, USA.

The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase eta (Poleta), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Poleta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Poleta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Poleta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Poleta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Poleta in replicating through D loop and DNA fragile sites.
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http://dx.doi.org/10.1038/nature09196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2899710PMC
June 2010