39 research outputs found
Biochemical Characterization of the WRN-1 RecQ Helicase of <i>Caenorhabditis elegans</i>
The highly conserved RecQ helicases are essential for the maintenance of genomic stability. Werner syndrome protein, WRN, is one of five human RecQ helicase homologues, and a deficiency of the protein causes a hereditary premature aging disorder that is characterized by genomic instability. A WRN orthologue, <i>wrn-1</i> lacking the exonuclease domain, has been identified in the nematode <i>Caenorhabditis elegans</i>. <i>wrn-1</i>(RNAi) in <i>C. elegans</i> has a shortened life span, increased sensitivity to DNA damage, and accelerated aging phenotypes. However, little is known about its enzymatic activity. We purified the recombinant <i>C. elegans</i> WRN-1 protein (CeWRN-1) and then investigated its substrate specificity in vitro to improve our understanding of its function in vivo. We found that CeWRN-1 is an ATP-dependent 3′−5′ helicase capable of unwinding a variety of DNA structures such as forked duplexes, Holliday junctions, bubble substrates, D-loops, and flap duplexes, and 3′-tailed duplex substrates. Distinctly, CeWRN-1 is able to unwind a long forked duplex compared to human WRN. Furthermore, CeWRN-1 helicase activity on a long DNA duplex is stimulated by <i>C. elegans</i> replication protein A (CeRPA) that is shown to interact with CeWRN-1 by a dot blot. The ability of CeWRN-1 to unwind these DNA structures may improve the access for DNA repair and replication proteins that are important for preventing the accumulation of abnormal structures, contributing to genomic stability
Radiometric DNA helicase assay using gap substrates with WRN in the presence of RPA.
<p>(A) Lanes 3–5 contained WRN (3 nM) and a range of RPA (6 nM, 12 nM, and 24 nM) concentrations with 3-nt gap substrates. Lane 2 contained only WRN (3 nM) and lane 6 contained only RPA (24 nM). Lane 1 contained control DNA substrates without any proteins and the substrates were boiled (lane7). (B) The reaction mixtures contained DNA substrate as indicated in the upper right hand corner of the figure which is exactly the same substrate shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004673#pone-0004673-g001" target="_blank">Figure 1B[c]</a> except for lacking the 3′-ss tail oligo. Lane 1 was a control reaction with only DNA substrate. Lane 7 was a heat-denatured substrate. Lane 2 was a control sample with only WRN. Lane 6 was a control samples with only RPA in the concentration range of 24 nM. Lanes 3–5 were reaction mixtures containing both RPA and WRN. WRN concentration was held constant at 3 nM and the RPA concentration was in the range of 6 nM to 24 nM. At the end of the enzymatic reaction, a cold-trap” oligomer complementary to the unlabeled, unwound oligomer was added. The arrows indicate the migration pattern of the unwound product (radiolabeled top strand oligomers). Asterisk indicates a 5′-<sup>32</sup>P labeled strand. Δ indicates boiled substrate.</p
The model of unique WRN helicase mechanism in BER or LP-BER.
<p>(A) Preceding steps in the BER or LP-BER involving glycosylases and APE1 result in gaps in the translocating and non-translocating DNA strands. WRN is capable of unwinding 1- to 2-nt gaps in the translocating strand proficiently and assists the strand displacement synthesis of polymerase β during subsequent steps. (B) The fork could regress into a Holliday junction (also called a chicken foot), where the nascent leading strand serves as a template for the lagging strand synthesis.</p
Radiometric DNA helicase assay using gap substrates and WRN.
<p>Each assay was performed using 1.5 nM of each substrate and a range of WRN concentration from 0.75 to 6 nM. A cold trap oligomer complementary to the unlabeled, unwound oligomer (100-fold) was added at the end of the enzymatic reaction. (A) A representative gel picture of the assay using 1-nt gap. The arrows indicate the migration pattern of the unwound product (radiolabeled ss oligomer). (B) A representative gel picture of the assay using 2-nt gap substrate. (C) A representative gel picture of the assay using 3-nt gap substrate. (D) A representative gel picture of the assay using DNA substrate without a gap. (E) Quantitations of at least three or two independent experiments shown in A and B. The graph is plotted as the percentage of unwound product on the Y-axis and the concentration of WRN on the X-axis. The mean values of three different experiments were plotted with standard deviation indicated by <i>error bars</i> (1-nt gap substrate and 2-nt gap substrate). Filled-circles indicate DNA substrate without a gap. Likewise, filled-squares indicate 1-nt gap substrate, filled-inverse triangles indicate 2-nt gap substrate and open triangles, 3-nt gap substrate. Asterisk indicates a 5′-<sup>32</sup>P labeled strand. Δ indicates boiled substrate.</p
Radiometric DNA helicase assay using WRN in the presence of POT1.
<p>The concentration range of POT1 variants used in these reactions was from 6 to 48 nM. The concentration of WRN-E84A was held constant at 6 nM. The DNA substrate used for these reactions is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004673#pone-0004673-g001" target="_blank">Figure 1B[d]</a>. (A) The reaction mixtures contained POT1v1 and WRN-E84A with the DNA substrate for POT1v1 to pre-bind. (B) The reaction mixtures contained POT1v2 and WRN-E84A with the same DNA substrate as (A). (C) Quantitations of at least two or more independent experiments shown in (A) is plotted. The graph is plotted as the percent of displacement of the radiolabeled oligomer on the Y-axis and the concentration of POT1 variants on the X-axis. Filled diamond represents reaction mixtures containing POT1v1 and WRN-E84A. Filled circle represents reaction mixture containing only POT1v1 and Filled triangle only WRN-E84A. (D) The reaction mixtures contained TRF2 and WRN-E84A with DNA substrate for TRF2 to pre-bind as shown in 1B[c]. TRF2 was added in these reactions from 12 nM (lane 4), 25 nM (lane 3), 50 nM (lane 2) to 100 nM (lane 1) and the concentration of WRN-E84A was held constant at 6 nM. Asterisk indicates a 5′-<sup>32</sup>P labeled strand. Δ indicates boiled substrate.</p
Radiometric DNA helicase assay using gap substrates and UvrD.
<p>The equal molar concentration range of UvrD was used from 0.75, 1.5, 3 to 6 nM. The arrows indicate the migration pattern of the unwound product (radiolabeled ss oligomer). (A) A representative gel picture of the assay using 1-nt gap. (B) A representative gel picture of the assay using 3-nt gap substrate. (C) Quantitations of at least two or more independent experiments shown in A and B. The graph is plotted as the percentage of unwound product on the Y-axis and the concentration of WRN on the X-axis. The mean values of replicate experiments were plotted with standard deviation indicated by <i>error bars</i>. Likewise, filled-diamond indicate 1-nt gap substrate, filled-triangle indicate 3-nt gap substrate. Asterisk indicates a 5′-<sup>32</sup>P labeled strand. Δ indicates boiled substrate.</p
A schematic diagram of recombinant proteins and DNA substrates.
<p>(A) The modulations of active helicases were studied using WRN variants. WRN-E84A is a full-length WRN with the N-terminal exonuclease inactivating mutation at E84 to alanine. WRN-HR is a WRN fragment containing helicase and RQC domains. (B) All DNA substrates contained 5′-10-nt poly-T tails while 3′-10-nt poly-T tails were excluded only in few of the indicated experiments. [a] Fork substrate without gap. [b] Fork substrate with 1 to 3-nt gap. [c] Fork substrate with 4 telomeric repeats located in the duplex DNA following 3-nt gap. [d] Fork substrate with 5 telomeric repeats located in ssDNA of the non-translocating strand.</p
Sequences of oligomers used for the making of DNA substrates.
<p>Sequences of oligomers used for the making of DNA substrates.</p
Site-Specific Noncovalent Interaction of the Biopolymer Poly(ADP-ribose) with the Werner Syndrome Protein Regulates Protein Functions
Werner syndrome is a premature aging disorder that is
caused by
defects in the Werner protein (WRN). WRN is a member of the RecQ helicase
family and possesses helicase and exonuclease activities. It is involved
in various aspects of DNA metabolism such as DNA repair, telomere
maintenance, and replication. Poly(ADP-ribose) polymerase 1 (PARP1)
is also involved in these processes by catalyzing the formation of
the nucleic-acid-like biopolymer poly(ADP-ribose) (PAR). It was previously
shown that WRN interacts with PARP1 and that WRN activity is inhibited
by PARP1. Using several bioanalytical approaches, here we demonstrate
that the enzymatic product of PARP1, <i>i.e.</i>, PAR, directly
interacts with WRN physically and functionally. First, WRN binds HPLC-size-fractionated
short and long PAR in a noncovalent manner. Second, we identified
and characterized a PAR-binding motif (PBM) within the WRN sequence
and showed that several basic and hydrophobic amino acids are of critical
importance for mediating the PAR binding. Third, PAR-binding inhibits
the DNA-binding, the helicase and the exonuclease activities of WRN
in a concentration-dependent manner. On the basis of our results we
propose that the transient nature of PAR produced by living cells
would provide a versatile and swiftly reacting control system for
WRN’s function. More generally, our work underscores the important
role of noncovalent PAR-protein interactions as a regulatory mechanism
of protein function
NCL inhibits WRN unwinding of G4 tetraplex DNA.
<p>The preparation of G4 tetraplex substrate was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035229#s2" target="_blank">Materials and Methods</a>. Purified WRNp (5 fmol) was incubated with 40 fmol G4 DNA substrate and 125 or 200 fmol ΔN-NCL (lanes 9–10) or 40, 125 or 200 fmol RGG fragment (lanes 15–17). Other lanes contain controls-ΔN-only ΔN-NCL (40 fmol, lane 3), 1-1-RBD 1–2 fragment (200 fmol, lanes 4, 11and 12), 3-4-RBD 3–4 fragment (200 fmol, lanes 5, 13 and 14), GST- GST protein (200 fmol, lane 19), Only WRN protein on lanes 8 and 18, B-only reaction buffer (lane 2), Δ- heat denatured substrate (lane 1). Reactions were terminated after 20 min at 37°C and run out on 8% polyacrylamide gels. A representative intact gel is shown.</p