The Werner Syndrome Gene Product Co-purifies with the DNA Replication Complex and Interacts with PCNA and Topoisomerase I

Abstract

Werner syndrome (WS) is a recessive disorder characterized by genomic instability and by the premature onset of a number of age-related diseases. To understand the molecular basis of this disease, we deleted a segment of the murine Wrn gene and created Wrn-deficient embryonic stem (ES) cells. At the molecular level, wild type-but not mutant-WS protein co-purifies through a series of centrifugation, chromatography, and sucrose gradient steps with the well characterized 17 S multiprotein DNA replication complex. Furthermore, wild type WS protein co-immunoprecipitates with a prominent component of the multiprotein replication complex, proliferating cell nuclear antigen (PCNA). In vitro studies also indicate that PCNA binds to a region in the N terminus portion of the WS protein containing a potential 3-5 exonuclease domain. Finally, human WS protein also co-immunoprecipitates with both PCNA and topoisomerase I. These results suggest that the WS protein interacts with several components of the DNA replication fork.Werner syndrome (WS) 1 is a rare disorder characterized by the premature onset of a number of processes associated with aging (1, 2). In addition, the proliferative life span of WS fibroblasts is reduced compared with age-matched controls (3)(4)(5). WS cells from patients also exhibit genomic abnormalities such as variegated chromosomal translocations and deletions (6, 7). Most recently, WS cells have been shown to have an attenuation of a p53-dependent apoptotic pathway (8).The WS gene (WRN) product contains seven helicase consensus domains that are 34 -38% identical to the Escherichia coli RecQ gene (9,10) and to the putative yeast helicase Sgs1p (11,12). The Sgs1p is known to interact with types I and II topoisomerases and is required for genome stability in Saccharomyces cerevisiae (11)(12)(13). In addition, an interesting model involving the promotion of extrachromosomal rDNA circles has been proposed for the aging phenotype in yeast Sgs1 mutants (14). Several studies suggest that the WS protein may be implicated in some aspect of DNA replication. WS cells show a prolongation of the S phase of the cell cycle and a decreased rate of DNA synthesis (15). Interestingly, the homologue of the human Werner syndrome gene product in Xenopus laevis is required for the formation of replication foci in egg extracts (16).To study the possible interaction of the mouse WS protein with the DNA replication apparatus, we recently created a deletion of part of the helicase domain of the murine homologue of the WS gene in embryonic stem (ES) cells (17). Biochemical studies of our ES cell lines allowed us to conclude that the WS protein co-purifies with the DNA replication complex and binds to certain of its specific components. EXPERIMENTAL PROCEDURESProtein Analysis-Generation and maintenance of the wild type and homozygous mutant embryonic stem cells have been described previously (17). Protein extraction, immunoprecipitations, and Western blotting analyses were performed as described (8,18). A rabbit polyclonal anti...

The role of DNA exonucleases in protecting genome stability and their impact on ageing

“…This is supported by the accumulation of stalled replication forks in cells lacking WRN protein (Rodriguez-Lopez et al 2002;Sidorova et al 2008), and it is likely that this reflects a need for the nuclease activity of WRN, since many of the defects observed in WS cells, including reduced proliferative capacity, low S-phase fraction and drug sensitivity, can be corrected by ectopic expression of a bacterial Holliday junction resolvase (Rodriguez-Lopez et al 2007). Recruitment of WRN to stalled replication forks may occur through binding to PCNA via a classical PIP (Lebel et al 1999;Rodriguez-Lopez et al 2003).…”

Vitamin C restores healthy aging in a mouse model for Werner syndrome

“…The reason for the difference between Wrn-null and Wrn Δhel/Δhel mice is unknown. Although we cannot rule out the possibility that a DNA helicase mutant Wrn protein could act as a dominant-negative peptide, this Wrn mutant protein does not associate with the DNA replication complex like the normal protein (61). Experiments are currently under way to determine whether the helicase mutant Wrn protein can interact and affect the enzymatic activity of known Wrn protein partners.…”

Deficient DNA repair in the human progeroid disorder, Werner syndrome

“…Recently it has been shown that WRN knockdown cells are hypersensitive to the methylating agents methyllexitropsin and temozolomide compared to isogenic controls [25]. In addition, WRN has been shown to physically interact with several proteins involved in SSBR/BER including: DNA polymerase ␦ (pol ␦) [26], DNA polymerase ␤ (pol ␤) [27], proliferating cell nuclear antigen (PCNA) [28], replication protein A (RPA) [29], flap endonuclease 1 (FEN-1) [30], and poly(ADPribose) polymerase 1 (PARP-1) [31]. WRN has been shown to stimulate FEN-1 flap cleavage [30] and nucleotide incorporation by pol ␦ [32].…”