The Werner syndrome protein binds replication fork and holliday junction DNAs as an oligomer

Abstract

Werner syndrome is an inherited disease displaying a premature aging phenotype. The gene mutated in Werner syndrome encodes both a 3' --> 5' DNA helicase and a 3' --> 5' DNA exonuclease. Both WRN helicase and exonuclease preferentially utilize DNA substrates containing alternate secondary structures. By virtue of its ability to resolve such DNA structures, WRN is postulated to prevent the stalling and collapse of replication forks that encounter damaged DNA. Using electron microscopy, we visualized the binding of full-length WRN to DNA templates containing replication forks and Holliday junctions, intermediates observed during DNA replication and recombination, respectively. We show that both wild-type WRN and a helicase-defective mutant bind with exceptionally high specificity (>1000-fold) to DNA secondary structures at the replication fork and at Holliday junctions. Little or no binding is observed elsewhere on the DNA molecules. Calculations of the molecular weight of full-length WRN revealed that, in solution, WRN exists predominantly as a dimer. However, WRN bound to DNA is larger; the mass is consistent with that of a tetramer.

FIGURE 1.

Binding of WRN to Holliday junction DNA. Wild-type (A) or K577M (B) WRN was incubated with Holliday junction templates, mounted onto carbon-coated copper grids, and rotary shadowcast with tungsten for visualization by EM. Images are shown in reverse contrast. Bar is equivalent to 50 nm.

FIGURE 2.

Binding of WRN to replication fork DNA. Wild-type (A) or K577M (B) WRN was incubated with replication fork DNA templates and processed for EM as described in the legend to Fig. 1. Images are shown in reverse contrast. Bar is equivalent to 100 nm.

FIGURE 3.

Quantitative analysis of WRN binding to Holliday junctions and replication forks. Large numbers of DNA molecules were surveyed to determine protein-free and protein-bound fractions. The protein-bound fractions were scored for specific and nonspecific binding (defined respectively, as junction binding or binding elsewhere on the DNA). A and C, percent Holliday junction molecules bound by wild-type (n = 359) and K577M-WRN (n = 527), respectively. B and D, percent replication forks bound by wild-type (n = 230) and K577M-WRN (n = 237), respectively. Error bars represent ± S.D.

FIGURE 4.

Size analysis of free and DNA-bound WRN proteins. Wild-type (top panel) and K577M (middle panel) WRN were bound to Holliday junctions (A, B, E, and F) and replication forks (C, D, G, and H) and were mounted side-by-side with apoferritin (K) on separate grids for EM analysis along with unbound wild-type (I) and K577M-WRN (J). The projected areas of wild-type and K577M-WRN were measured from EM images and used to estimate the oligomeric forms of free and DNA-bound WRN as described under “Experimental Procedures.”

FIGURE 5.

Distribution of WT-WRN and K577M-WRN areas measured by EM. Projected areas of free and DNA-bound wild-type and K577M-WRN and apoferritin were calculated from EM images as described under “Experimental Procedures.” RF and HJ refer to replication fork DNA and Holliday junction DNA, respectively.