Abstract
Werner syndrome is a premature aging and cancer-prone hereditary disorder caused by deficiency of the WRN protein that harbors 3' -->5' exonuclease and RecQ-type 3' --> 5' helicase activities. To assess the possibility that WRN acts on partially melted DNA intermediates, we constructed a substrate containing a 21-nucleotide noncomplementary region asymmetrically positioned within a duplex DNA fragment. Purified WRN shows an extremely efficient exonuclease activity directed at both blunt ends of this substrate, whereas no activity is observed on a fully duplex substrate. High affinity binding of full-length WRN protects an area surrounding the melted region of the substrate from DNase I digestion. ATP binding stimulates but is not required for WRN binding to this region. Thus, binding of WRN to the melted region underlies the efficient exonuclease activity directed at the nearby ends. In contrast, a WRN deletion mutant containing only the functional exonuclease domain does not detectably bind or degrade this substrate. These experiments indicate a bipartite structure and function for WRN, and we propose a model by which its DNA binding, helicase, and exonuclease activities function coordinately in DNA metabolism. These studies also suggest that partially unwound or noncomplementary regions of DNA could be physiological targets for WRN.
Highlights
Syndrome (WS)1 phenotype is characterized by early onset of a number of aging characteristics, including graying and loss of hair, increased wrinkling and ulceration of the skin, osteoporosis, atherosclerosis, and an increased frequency of age-related maladies such as cancer, diabetes, and cataracts
We show that ATP hydrolysis is not required for high affinity binding of WRN to the melted region, and that the exonuclease domain does not bind stably to this substrate
Genomic instability in the premature aging and cancerprone hereditary disease, WS, is thought to be caused by DNA metabolic defects that result from a lack of WRN protein
Summary
WRN Purification—Recombinant wild type and mutant WRN proteins were overexpressed and purified essentially as described previously [29]. Electrophoretic Mobility Shift Assay—Fully duplex or bubble-containing substrate (32P-labeled, 0.5 fmol) was incubated with wild type WRN (12–72 fmol) or mock protein preparation (volume normalized) for 30 min at 4 °C in binding buffer (10 l final volume) containing 20 mM HEPES-KOH (pH 8.0), 1 mM MgCl2, 0.1 mM EDTA, 100 g/ml BSA, 0.5 mM dithiothreitol, 0.1% Nonidet P-40, 5% glycerol, 4% Ficoll, and 1 mM ATP␥S or ATP. DNase I Footprinting—32P-Labeled DNA substrates (21-nt bubble or completely complementary, 0.5 fmol each) were incubated with or without WRN wild type or mutant protein (WRNp, WRN-E84A, WRNK577M, or WRN⌬369 –1432) for 5–30 min at 4 °C or 37 °C in WRN reaction buffer (10-l final volume). The digestion products of these reactions were resolved by denaturing polyacrylamide (14%) gel electrophoresis in 1ϫ
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