Werner Syndrome Protein

Abstract

In addition to its DNA helicase activity, Werner syndrome protein (WRN) also possesses an exonuclease activity (Shen, J.-C. , Gray, M. D., Kamath-Loeb, A. S., Fry, M., Oshima, J., and Loeb, L. A. (1998) J. Biol. Chem. 273, 34139 -34144). Here we describe the properties of nearly homogeneous WRN exonuclease. WRN exonuclease hydrolyzes a recessed strand in a partial DNA duplex but does not significantly digest single-stranded DNA, blunt-ended duplex, or a protruding strand of a partial duplex. Although DNA is hydrolyzed in the absence of nucleoside triphosphates, nuclease activity is markedly stimulated by ATP, dATP, or CTP. WRN exonuclease digests DNA with a 3 3 5 directionality to generate 5-dNMP products, and DNA strands terminating with either a 3-OH or 3-PO 4 group are hydrolyzed to similar extents. A recessed DNA strand with a single 3-terminal mismatch is hydrolyzed more efficiently by WRN than one with a complementary nucleotide, but the enzyme fails to hydrolyze a DNA strand terminating with two mismatched bases. WRN exonuclease is distinguished from known mammalian DNA nucleases by its covalent association with a DNA helicase, preference for a recessed DNA strand, stimulation by ATP, ability to equally digest DNA with 3-OH or 3-PO 4 termini, and its preferential digestion of DNA with a single 3-terminal mismatch.Werner Syndrome (WS) 1 is a recessive inherited disease characterized by genetic instability and aging in early adulthood (1, 2). The gene defective in WS, WRN, encodes a 3Ј 3 5Ј RecQ-like DNA helicase that unwinds DNA in an ATP-dependent manner (3-5). Mutations in WRN are invariably found in patients exhibiting the clinical symptoms of WS (6, 7). These include atherosclerosis, osteoporosis, diabetes mellitus, and bilateral cataracts, as well as an unusually high incidence of tumors of non-epithelial cell origin. At the cellular level, WS cells are characterized by chromosomal translocations, large DNA deletions, elevated rates of homologous recombination, defective maintenance of telomeres, and a prolonged S-phase of DNA synthesis (8 -15).In the preceding paper (16), we reported the identification of a novel exonuclease activity in WRN. We used molecular genetic, biochemical, and immunochemical methods to establish that the exonuclease, like the DNA helicase, is integral to WRN. Although the two activities are expressed in the same polypeptide in the wild-type protein, they can be uncoupled from each other by introducing mutations separately in each of the two domains. In patients, mutations in WRN are not necessarily located in the helicase domain. They are found throughout the gene and invariably introduce stop codons or deletions (6, 7). It has been argued that many mutations obliterate the nuclear localization signal and that lack of localization may be important in the pathogenesis of WS (17, 18). This lack of nuclear localization would result in deficits of both helicase and exonuclease activities.To gain a better understanding of the functions of the WRN exonuclease, we have studied its...

Biochemical and Kinetic Characterization of the DNA Helicase and Exonuclease Activities of Werner Syndrome Protein

“…The DNA replication phenotypes of Werner syndrome cells (12)(13)(14) suggest that WRN has a role in DNA synthesis. WRN preferentially digests DNA with a single 3Ј terminal mismatch (29), suggesting that the 3Ј to 5Ј exonuclease activity of WRN may remove a mismatched nucleotide that was incorporated by a DNA polymerase. The results presented here indicate that Zn 2ϩ is an important metal cofactor for WRN exonuclease activity.…”

“…WRN, like all other DNA helicases characterized to date, utilizes the energy from nucleotide hydrolysis to unwind double-stranded DNA (25)(26)(27)(28). Although the nucleotide preference of WRN helicase and exonuclease activities has been examined (29,30), little is known about the optimal solution conditions for WRN catalytic activities. Recent work from the Kowalczykowski laboratory (31) demonstrated that E. coli RecQ helicase activity is sensitive to the ratio of magnesium ion to ATP concentration with an optimal ratio of 0.8 and a free magnesium ion concentration of 50 M. In addition, E. coli RecQ helicase activity displayed a sigmoidal dependence on ATP concentration, suggesting multiple interacting ATP sites (31).…”

WRN helicase accelerates the transcription of ribosomal RNA as a component of an RNA polymerase I-associated complex

“…In addition, advanced sequence alignment analysis revealed a putative exonuclease domain near the N-terminus of WRN (Mushegian et al, 1997). Indeed, an exonuclease activity of puri®ed WRN protein has been demonstrated (Huang et al, 1998;Kamath-Loeb et al, 1998;Shen et al, 1998;Suzuki et al, 1999).…”

Regulation and mechanisms of mammalian double-strand break repair

“…WRN (Kamath-Loeb et al, 1998;Cooper et al, 2000;Li and Comai, 2000) and MRE11 Trujillo et al, 1998) are 3 0 -5 0 exonucleases with a preference for recessed 3 0 ends, and WRN is stimulated by KU (Cooper et al, 2000;Li and Comai, 2001) but inhibited by DNA-PKcs . WRN can displace DNA-PKcs from DNA-PK holoenzyme bound to a DNA end (Li and Comai, 2002).…”

“…On the other hand, the WRN helicase/exonuclease is implicated by virtue of its interaction with and stimulation by KU (Cooper et al, 2000;Li and Comai, 2001). DNase III, which is the dominant 3 0 -5 0 exonuclease in mammalian cell extracts, may also play a role, as it can digest 3 0 overhangs (Hoss et al, 1999;Mazur and Perrino, 1999), which are relatively resistant to MRE11 and WRN (Kamath-Loeb et al, 1998;Cooper et al, 2000;Huang et al, 2000;Li and Comai, 2000). Notably, however, a number of in vitro and in vivo studies suggest that microhomology-based SSA, unlike accurate end-joining, can occur in the absence of KU (Gottlich et al, 1998;Kabotyanski et al, 1998;Chen S et al, 2001).…”