Probing the Roles of Active Site Residues in the 3′-5′ Exonuclease of the Werner Syndrome Protein

Abstract

Werner syndrome is a human autosomal recessive disorder and is associated with premature aging and an increased incidence of cancer (1, 2). Werner syndrome patients exhibit an increased predisposition to arteriosclerosis, osteoporosis, type II diabetes mellitus, and a variety of tumors, which are normally observed during aging (2, 3). Fibroblast cultures from Werner syndrome patients show a reduced life span and a variety of chromosomal abnormalities including reciprocal translocations, deletions, and inversions (4), abnormalities in S phase initiation or transit (5), and attenuated replicative potential (6, 7). In addition, some Werner syndrome cell lines have shown aberrant mitotic recombination (8) and increased levels of homologous recombination (9). Werner syndrome cell lines are also hypersensitive to the DNA-damaging agent, 4-nitroquinoline-1-oxide (10). Together, these cellular phenotypes suggest that Werner syndrome is associated with one or more defects in DNA metabolism.Most Werner syndrome cases have been causally related to mutations in a single gene, WRN, which encodes a multifunctional protein, WRN, a member of the RecQ family of helicases (11). In humans, this family includes proteins such as Bloom syndrome and Rothmund-Thompson syndrome proteins, whose germline mutations are responsible for diseases associated with genomic instability (12, 13).The precise molecular function of WRN is unclear. However, WRN is known to interact with several key proteins that play critical roles in DNA replication, recombination, and repair, which suggests that WRN may maintain genomic integrity and life span by participating in DNA transactions in mammalian cells (13).WRN contains an exonuclease domain that is unique among RecQ family members (14 -16). Although the biological importance of the Werner exonuclease domain (WRNexo) 3 has not been precisely defined, the facts that WRNexo cleaves diverse substrates including the long fork form of DNA and Holliday junctions, and that WRN interacts with proliferating cell nuclear antigen (PCNA) suggest that WRNexo may participate in the DNA replication process (16). Recent studies show that inactivation of WRNexo alters DNA end-joining in human cells (17). Moreover, Ku70/80, an important regulator of genomic stability, stimulates the exonuclease activity of WRNexo, implying that WRNexo may play an important role in DNA repair (17). Interestingly, Ku70/80 inhibits the 3Ј-5Ј exonuclease of Klenow fragment (KFexo), a structurally conserved 3Ј-5Ј exonuclease, which suggests that the Ku-mediated activation mechanism could be unique to WRNexo. Thus, elucidation of structure and function of WRNexo may provide important insights to understand the molecular mechanism by which * This work was supported by funds from the National Creative Research Initiatives (Ministry of Science and Technology). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Sect...

The Werner Syndrome Helicase/Exonuclease Processes Mobile D-Loops through Branch Migration and Degradation

“…In order to examine branch migration activity of the wild type WRN protein preparation in the absence of digestion, we suppressed the WRN exonuclease activity by decreasing the free Mg 2+ concentration. Since the WRN exonuclease acts by a two-metal ion dependent mechanism, free divalent cations are required to bind the exonuclease active site [43]–[45]. While WRN helicase remains active at Mg 2+ ∶ATP ratios near 1 [43](data not shown), we found that WRN exonuclease activity is highly dependent on the free Mg 2+ concentration.…”

WRN Exonuclease activity is blocked by specific oxidatively induced base lesions positioned in either DNA strand

“…Our results suggest that WRN interacts with the non-digested strand when degrading the opposite strand, consistent with the ring model of WRN exonuclease proposed by Perry et al (24), where both DNA strands appear to contact the exonuclease domain. Furthermore, the mouse WRN exonuclease appears to work as a multimer (65). As there is no information currently available regarding the structure of WRN exonuclease binding longer double-stranded DNA substrates, more studies are required to fully explain our results.…”

Synchrotron Radiation Facilities in Korea: Pohang Light Source and Future XFEL Project

“…Solar cells were made of composites of poly(3-hexylthiophene) (P3HT) and a fullerene derivative and their high performance was found to depend on the structure and orientation of the P3HT matrix through detailed GIXS analysis [9]. Furthermore, SAXS studies were extended for the structural analysis of proteins and polynucleic acids in various conditions including in nature and complexes with diverse ligands including drugs [10][11][12][13][14].…”