A Novel Protein Interacts with the Werner's Syndrome Gene Product Physically and Functionally

Abstract

Werner's syndrome (WS) 1 is a rare autosomal recessive disorder characterized by premature aging and an early onset of age-related diseases including arteriosclerosis, malignant neoplasms, melituria, and cataract (1). Somatic cells derived from WS patients show chromosome instability, a shorter life span in in vitro culture, and accelerated telomere shortening (2, 3). WS cells have subtle defects in DNA replication, resulting in a reduced frequency of firing of replication origins (4). In addition, a large number of reports have shown that many cellular events including DNA repair, transcription, and apoptosis are affected in WS cells (5-7). The gene responsible for WS encodes a protein (WRN) that is a member of the RecQ family of DNA helicases (8). Most of the WS mutations that have been identified are nonsense or frameshift mutations, resulting in the truncation of WRN (9, 10). The clinical features and cellular phenotypes of most WS patients seem to be due to an absolute lack of WRN in the nucleus because the nuclear localization signal of WRN resides in its C-terminal end (11).The RecQ family includes Escherichia coli RecQ, S. cerevisiae Sgs1, Shizosaccharomyces pombe Rqh1, and five human RecQ helicases, namely DNA helicase Q1/RecQL (RecQL1), WRN, Bloom's syndrome gene product (BLM), Rhusmund-Thomson's syndrome gene product (RecQL4), and RecQL5 (12-19). Rhusmund-Thomson's syndrome also shows some features of the premature aging phenotype, and Bloom's syndrome is characterized by a predisposition to various malignant neoplasms. In S. cerevisiae, mutations in the SGS1 gene caused premature aging and hyper-recombination phenotypes (20, 21). The sgs1 mutants showed higher sensitivity to MMS and hydroxyurea (22)(23)(24)(25)37). Thus, sgs1 mutants exhibit some of the phenotypes of WS.WRN has been shown to have DNA helicase and exonuclease activity (26 -29). Recent studies (30 -32) have revealed that WRN interacts with replication protein A, PCNA, DNA topoisomerase I, and DNA polymerase ␦, indicating the involvement of WRN in some aspects of DNA replication. WRN also interacts with the p53 and Ku 70/86 heterodimer, suggesting that WRN is involved in apoptosis and the repair of DNA double strand breaks (7,(33)(34)(35). Despite these observations, it is not clear how the dysfunction of WRN is related to the observed phenotypes of WS cells. To obtain further insight into the process in which WRN is involved, we performed a two-hybrid screening using mouse WRN (mWRN) as bait and identified three interacting proteins: Ubc9, SUMO-1 (small ubiquitinrelated modifier-1), and a novel protein, WHIP (Werner Helicase Interacting Protein), which is conserved from E. coli to human (36). Here we report that mWRN physically interacts with mWHIP, and the yeast homologue of WRN, Sgs1, genetically interacts with yWHIP. EXPERIMENTAL PROCEDURESTwo-hybrid Assay-The yeast strains and plasmids for two-hybrid screening were described previously (36).

A mutation in EXO1 defines separable roles in DNA mismatch repair and post-replication repair

“…MGS1 encodes for a DNA-dependent ATPase with ssDNA annealing activities that has been suggested to compete with the PRR pathway for resolution of fork stalling lesions [6,37,38]. RAD30 has been implicated in an error-free form of translesion synthesis [39,40] that some have placed in the PRR pathway [41].…”

Werner Helicase-interacting Protein 1 Binds Polyubiquitin via Its Zinc Finger Domain

“…Annotated domains in WRNIP1 include a small Rad18 (radiation-sensitive 18)-like zinc finger at the N terminus and a AAAϩ ATPase domain in the middle of the protein. The homology of WRNIP1 with the replication factor C family of clamp loader proteins, along with its ability to stimulate DNA polymerase ␦ synthesis of new DNA, suggests a possible role for WRNIP1 in replication and/or replication-dependent DNA repair (18,19).…”

Werner Interacting Protein 1 Promotes Binding of Werner Protein to Template-Primer DNA

“…10,11) Genetic analyses using MGS1 mutants revealed that Mgs1p is required to prevent genome instability caused by replication arrest and is not involved in the repair of DNA lesions.…”

“…15) In this context, it is of note that WRN interacts with Pold 16) and stimulates the DNA synthesizing activity of Pold by increasing the length of synthesized DNA. 17) Since WRNIP1 interacts with WRN, 10,18) WRNIP1 and WRN may interact with templateprimer DNA in a cooperative fashion to regulate the activity of Pold. However, the biochemical relationships between WRNIP1 and WRN at the template-primer DNA remain elusive.…”

“…10,11) Genetic analyses using MGS1 mutants revealed that Mgs1p is required to prevent genome instability caused by replication arrest and is not involved in the repair of DNA lesions. 12) In addition, overproduction of Mgs1p is lethal or very toxic when it is combined with mutations in the genes that encode proteins involved in DNA replication, such as replication protein A, replication factor C, proliferating cell nuclear antigen and DNA polymerase d (Pold).…”