The Werner Syndrome Protein Binds Replication Fork and Holliday Junction DNAs as an Oligomer

Sarah A. Compton,Lawrence A. Loeb,Jack D. Griffith,Ashwini S. Kamath-Loeb,Gökhan Tolun

doi:10.1074/jbc.m803370200

Sarah A. Compton, Lawrence A. Loeb + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m803370200

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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.

Highlights

Facilitate strand separation either passively, by binding and trapping ssDNA2 that arises during transient breathing of paired DNA, or by an active mode wherein they actively destabilize paired DNA in addition to binding the released ssDNA [2]
We present initial studies on the visualization of WRN binding to model DNA substrates encountered during DNA replication and recombination by electron microscopy (EM)
We show that WRN binds at the junction of a replication fork and at Holliday junction structures

Summary

EXPERIMENTAL PROCEDURES

Wild-type and K577M WRN Purification—WRN was purified as described by successive steps of DEAE-cellulose, phosphocellulose, and Ni2ϩ-affinity chromatography [9]. The Holliday junction was synthesized by annealing four oligonucleotides together resulting in a small four-way Holliday junction with AGCC-3Ј overhangs These junctions were transformed into larger Holliday junction templates suitable for visualization with EM by ligation of four 575-bp double strand DNA arms onto the junction. The construct was nicked with N.BbvCIA followed by strand displacement with Klenow (exo-) in the absence of dCTP This resulted in a circular template with a 400-bp single strand arm. Protein-DNA Binding Reactions and Preparation of Samples for EM—Wild-type or K577M WRN (0.8 ␮g/ml) was incubated with DNA (3 ␮g/ml) on ice in 30 ␮l of binding buffer containing 40 mM Tris-HCl (pH 7.6), 4 mM MgCl2, 5 mM dithiothreitol, and 1 mM ATP␥S for 20 min. Number of particles traced for size analysis The following abbreviations are used: WT, wild-type; RF, replication fork DNA; HJ, Holliday junction DNA

Free WRN

RESULTS

Apoferritin standards were crosslinked in parallel and mounted with

Dimer Tetramer

Findings

DISCUSSION