The DNA helicase activity of yeast Sgs1p is essential for normal lifespan but not for resistance to topoisomerase inhibitors

The Saccharomyces cerevisiae SGS1 gene is a member of the RecQ family of ATP-dependent DNA helicases, which includes the human WRN, BLM and RECQ4 genes. Mutations in the WRN gene cause the human premature ageing disorder, Werner's syndrome. Deletion of the SGS1 gene also causes premature ageing in yeast, suggesting that the molecular mechanisms of cellular ageing may be evolutionarily conserved. To investigate the role of the RecQ helicase domain in ageing, a point mutation (SGS1 K(706)-->A) known to eliminate the DNA helicase activity of Sgs1p was constructed. This mutant allele failed to rescue the premature ageing of the sgs1Delta strain, demonstrating that Sgs1p DNA helicase activity is required for a normal lifespan. In contrast, the SGS1 K(706)-->A allele was sufficient to rescue the hypersensitivity of the sgs1Delta strain to topoisomerase inhibitors, but not other genotoxic agents. These findings support the idea that Sgs1p fulfils multiple cellular functions, and that DNA helicase activity is dispensable for some of these (e.g. functional interaction with topoisomerases), but essential for others (e.g. longevity).     

Characterisation of the interaction between WRN,the helicase/exonuclease defective in progeroid Werner's syndrome,and an essential replication factor,PCNA

Ageing is linked to the accumulation of replicatively senescent cells. The best model system to date for studying human cellular ageing is the progeroid Werner's syndrome (WS), caused by a defect in WRN, a recQ-like helicase that also possesses exonuclease activity. In this paper, we characterise the interaction between WRN and an essential replication factor, PCNA. We show that wild-type WRN protein physically associates with PCNA at physiological protein concentrations in normal cells, while no association is seen in cells from patients with WS. We demonstrate co-localisation of WRN and PCNA at replication factories, show that PCNA binds to two distinct functional sites on WRN, and suggest a mechanism by which association between WRN and PCNA may be regulated in cells on DNA damage and during DNA replication.     

The role of WRN in DNA repair is affected by post-translational modifications

Werner syndrome (WS) is an autosomal recessive progeroid disease characterized by genomic instability. WRN gene encodes one of the RecQ helicase family proteins, WRN, which has ATPase, helicase, exonuclease and single stranded DNA annealing activities. There is accumulating evidence suggesting that WRN contributes to the maintenance of genomic integrity through its involvement in DNA repair, replication and recombination. The role of WRN in these pathways can be modulated by its post-translational modifications in response to DNA damage. Here, we review the functional consequences of post-translational modifications on WRN as well as specific DNA repair pathways where WRN is involved and discuss how these modifications affect DNA repair pathways.     

The N-terminal domain of the large subunit of human replication protein A binds to Werner syndrome protein and stimulates helicase activity

Werner syndrome (WS) is a recessive inherited human disease characterized by the early onset of aging. The gene mutated in WS encodes a DNA helicase that unwinds the double helical structure of DNA in the 3'-->5' direction as well as a 3'-->5' exonuclease. Our previous studies indicated that the activity of Werner syndrome helicase (WRN) could be stimulated by human replication protein A (hRPA), a heterotrimeric single-stranded DNA binding protein. We now localize the interaction between WRN and hRPA by measuring the stimulation of helicase activity and the binding of WRN by hRPA and its derivatives. The large subunit of hRPA (hRPA70) stimulates WRN helicase to the same extent as the hRPA heterotrimer, whereas the dimer of the two smaller subunits (hRPA 32.14) does not stimulate. By examining hRPA70 mutants with progressive deletions from either the C- or N-terminus, we found that the domain responsible for stimulation lies in the N-terminal half of the protein. By using enzyme-linked immunosorbent assay (ELISA) to examine physical interaction between WRN and the same deletion mutants, we found that the WRN-binding motif is located within amino acids 100-300 and overlaps with the single-stranded DNA binding domain (amino acids 150-450). We suggest that hRPA, by engaging in both protein-protein and protein-DNA interactions, facilitates unwinding events catalyzed by WRN helicase during DNA synthetic processes. These data should help further elucidation of the molecular mechanisms of genetic instability and premature aging phenotypes manifested by WS.     

ABCD syndrome is caused by a homozygous mutation in the EDNRB gene

ABCD syndrome is an autosomal recessive syndrome characterized by albinism, black lock, cell migration disorder of the neurocytes of the gut (Hirschsprung disease [HSCR]), and deafness. This phenotype clearly overlaps with the features of the Shah-Waardenburg syndrome, comprising sensorineural deafness; hypopigmentation of skin, hair, and irides; and HSCR. Therefore, we screened DNA of the index patient of the ABCD syndrome family for mutations in the endothelin B receptor (EDNRB) gene, a gene known to be involved in Shah-Waardenburg syndrome. A homozygous nonsense mutation in exon 3 (R201X) of the EDNRB gene was found. We therefore suggest that ABCD syndrome is not a separate entity, but an expression of Shah-Waardenburg syndrome.     

DNA helicase deficiencies associated with cancer predisposition and premature ageing disorders

Deficiency in a helicase of the RecQ family is found in at least three human genetic disorders associated with cancer predisposition and/or premature ageing. The RecQ helicases encoded by the BLM, WRN and RECQ4 genes are defective in Bloom's, Werner's and Rothmund-Thomson syndromes, respectively. Cells derived from individuals with these disorders in each case show inherent genomic instability. Recent studies have demonstrated direct interactions between these RecQ helicases and human nuclear proteins required for several aspects of chromosome maintenance, including p53, BRCA1, topoisomerase III, replication protein A and DNA polymerase delta. Here, we review this network of protein interactions, and the clues that they present regarding the potential roles of RecQ family members in DNA repair, replication and/or recombination pathways.     

WRN participates in translesion synthesis pathway through interaction with NBS1

Werner syndrome (WS), caused by mutation of the WRN gene, is an autosomal recessive disorder associated with premature aging and predisposition to cancer. WRN belongs to the RecQ DNA helicase family, members of which play a role in maintaining genomic stability. Here, we demonstrate that WRN rapidly forms discrete nuclear foci in an NBS1-dependent manner following DNA damage. NBS1 physically interacts with WRN through its FHA domain, which interaction is important for the phosphorylation of WRN. WRN subsequently forms DNA damage-dependent foci during the S phase, but not in the G1 phase. WS cells exhibit an increase in spontaneous focus formation of polη and Rad18, which are important for translesion synthesis (TLS). WRN also interacts with PCNA in the absence of DNA damage, but DNA damage induces the dissociation of PCNA from WRN, leading to the ubiquitination of PCNA, which is essential for TLS. This dissociation correlates with ATM/NBS1-dependent degradation of WRN. Moreover, WS cells show constitutive ubiquitination of PCNA and interaction between PCNA and Rad18 E3 ligase in the absence of DNA damage. Taken together, these results indicate that WRN participates in the TLS pathway to prevent genomic instability in an ATM/NBS1-dependent manner.     

Unwinding the molecular basis of the Werner syndrome

Werner syndrome (WS) is an autosomal recessive disease manifested by the premature onset of age-related phenotypes, including diseases such as atherosclerosis and cancer. This mimicry of normal aging with the possible exception of central nervous system manifestations has made it a focus of recent molecular studies on the pathophysiology of aging. In culture, cells obtained from patients with WS are genetically unstable, characterized by an increased frequency of nonclonal translocations and extensive DNA deletions. The WS gene product (WRN) is a DNA helicase belonging to the RecQ family, but is unique within this family in that it also contains an exonuclease activity. In addition to unwinding double-stranded DNA, WRN helicase is able to resolve aberrant DNA structures such as G4 tetraplexes, triplexes and 4-way junctions. Concordant with this structure-specificity, WRN exonuclease preferentially hydrolyzes alternative DNA that contains bubbles, extra-helical loops, 3-way junctions or 4-way junctions. WRN has been shown to bind to and/or functionally interact with other proteins, including replication protein A (RPA), proliferating cell nuclear antigen (PCNA), DNA topoisomerase I, Ku 86/70, DNA polymerase delta and p53. Each of these interacting proteins is involved in DNA transactions including those that resolve alternative DNA structures or repair DNA damage. The biochemical activities of WRN and the functions of WRN associated proteins suggest that in vivo WRN resolves DNA topological or structural aberrations that either occur during DNA metabolic processes such as recombination, replication and repair, or are the outcome of DNA damage.     

Werner helicase relocates into nuclear foci in response to DNA damaging agents and co-localizes with RPA and Rad51

BACKGROUND: Werner syndrome (WS) is an autosomal recessive disorder with many features of premature ageing. Cells derived from WS patients show genomic instability, aberrations in the S-phase and sensitivity to genotoxic agents. The gene responsible for WS (WRN) encodes a DNA helicase belonging to the RecQ helicase family. Although biochemical studies showed that the gene product of WRN (WRNp) interacts with proteins that participate in DNA metabolism, its precise biological function remains unclear. RESULTS: Using immunocytochemistry, we found that WRNp forms distinct nuclear foci in response to DNA damaging agents, including camptothecin (CPT), etoposide, 4-nitroquinolin-N-oxide and bleomycin. The presence of aphidicolin inhibited CPT-induced WRNp foci strongly but not bleomycin-induced foci. These WRNp foci overlapped with the foci of replication protein A (RPA) almost entirely and with the foci of Rad51 partially, implicating cooperative functions of these proteins in response to DNA damage. We also found that WRNp foci partially co-localize with sites of 5-bromo-2'-deoxy-uridine incorporation. CONCLUSIONS: These findings suggest that WRNp form nuclear foci in response to aberrant DNA structures, including DNA double-strand breaks and stalled replication forks. We propose that WRNp takes part in the homologous recombinational repair and in the processing of stalled replication forks.     

PCR assay confirms diagnosis in syndrome with variably expressed phenotype: mutation detection in Stickler syndrome.

Stickler syndrome is an autosomal dominant disease with ocular (severe myopia, vitreal degeneration, and retinal detachment) and other systemic manifestations (hearing loss, cleft palate, epiphyseal dysplasia, and premature osteoarthritis). As with other dominantly inherited conditions, the clinical phenotype of Stickler syndrome varies considerably. To date, all mutations have been located in the type II procollagen (COL2A1) gene. Analysis of a C-->T mutation we had identified previously, in COL2A1 gene in exon 40, in a three generation pedigree showed the loss of a cleavage site for the TaqI restriction enzyme. We designed a rapid PCR based restriction enzyme assay to detect this mutation and used it to establish the diagnosis in a neonate from the same pedigree, presenting with the first occurrence of the Pierre-Robin sequence in the family and minimal ocular findings. These results underline the potential diagnostic value of many as yet undetected DNA mutations in families affected with Stickler syndrome, since the variability of the phenotype can impede accurate diagnosis, appropriate genetic counselling, and effective intervention and prophylactic treatment for affected people.     

家族性腺瘤性息肉病家系中一种新的APC基因的胚系突变

目的研究1个家族性腺瘤性息肉病家系的腺瘤样息肉病基因（adenomatous polyposis coli，APC）的胚系突变。方法经结肠镜、组织病理学检查和家族史的调查，确定了1例家族性腺瘤性息肉病（familial adenomatous polyposis，FAP）患者。应用多重连接依赖性探针扩增（multiplex ligation-dependent probe amplification，MLPA）、变性高效液相色谱（denaturing high-performance liquid chromatography，DHPLC）测序等技术对这一家系的成员进行系统的APC全基因筛查。结果在此家系中发现一个新的APC基因的胚系突变c。1999 C〉T（Q667X），这一突变造成了APC基因终止密码子的形成，从而形成有功能障碍的截短蛋白。临床上，此突变可引起严重的FAP症状，早发结直肠腺瘤和腺癌。结论Q667X胚系突变是引起该家系临床表型的原因，受累成员可考虑大肠预防性切除手术。     

Werner syndrome and mutations of the WRN and LMNA genes in France

Werner syndrome (WS) is a pleiotropic disease of premature aging involving short stature, tight, atrophied, and/or ulcerated skin; a characteristic 'birdlike' facies and high, squeaky or hoarse voice; premature greying and thinning of the hair; and early onset cataracts. Additional common symptoms include diabetes mellitus, hypogonadism, osteoporosis, osteosclerosis of the digits, soft tissue calcification, premature atherosclerosis, rare or multiple neoplasms, malformed teeth, and flat feet. Diagnosis can be difficult due to the variable presentation and rarity of the disorder. Transmission is usually autosomal recessive. The WS gene, WRN, is member of the RecQ DNA helicase family. Biallelic mutations of WRN are responsible for most patients. Although heterozygous missense mutations in the LMNA gene have been observed in severely affected WS patients, this only accounts for a small fraction of non-WRN patients. Eighteen WS cases were referred to us for molecular analysis. Eleven had definite and three had probable WS according to the University of Washington Registry clinical criteria. All exons of the WRN gene and their splice junctions were sequenced. Of the fourteen definite or probable cases, 11 had one or more WRN mutation. Thirteen different mutations were found, and ten of these were previously undescribed. There were few phenotypic differences between patients with WRN mutation(s) and those who met clinical criteria though lacking WRN mutations. However, patients with mutations tended to have more symptoms overall, and mutations were not observed in the two cases with cardiomyopathy.     

Homozygous and compound heterozygous mutations at the Werner syndrome locus

The Werner syndrome (WS) is a rare autosomal recessive progeroid disorder. The Werner syndrome gene (WRN) has recently been identified as a member of the helicase family. Four distinct mutations were previously reported in three Japanese and one Syrian WS pedigrees. The latter mutation was originally described as a 4 bp deletion spanning a spliced junction. It is now shown that this mutation results in a 4 bp deletion at the beginning of an exon. Nine new WRN mutations in 10 additional WS patients, both Japanese and Caucasian, are described. These include three compound heterozygotes (one Japanese and two Caucasian). The new mutations are located all across the coding region.