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. 2012 Aug 31;287(36):30151-6.
doi: 10.1074/jbc.M112.389791. Epub 2012 Jul 11.

The Werner syndrome protein promotes CAG/CTG repeat stability by resolving large (CAG)(n)/(CTG)(n) hairpins

Nelson L S Chan  1 , Caixia Hou, Tianyi Zhang, Fenghua Yuan, Amrita Machwe, Jian Huang, David K Orren, Liya Gu, Guo-Min Li
Affiliations

The Werner syndrome protein promotes CAG/CTG repeat stability by resolving large (CAG)(n)/(CTG)(n) hairpins

Nelson L S Chan et al. J Biol Chem. .

Abstract

Expansion of CAG/CTG repeats causes certain neurological and neurodegenerative disorders, and the formation and subsequent persistence of stable DNA hairpins within these repeats are believed to contribute to CAG/CTG repeat instability. Human cells possess a DNA hairpin repair (HPR) pathway, which removes various (CAG)(n) and (CTG)(n) hairpins in a nick-directed and strand-specific manner. Interestingly, this HPR system processes a (CTG)(n) hairpin on the template DNA strand much less efficiently than a (CAG)(n) hairpin on the same strand (Hou, C., Chan, N. L., Gu, L., and Li, G. M. (2009) Incision-dependent and error-free repair of (CAG)(n)/(CTG)(n) hairpins in human cell extracts. Nat. Struct. Mol. Biol. 16, 869-875), suggesting the involvement of an additional component for (CTG)(n) HPR. To identify this activity, a functional in vitro HPR assay was used to screen partially purified HeLa nuclear fractions for their ability to stimulate (CTG)(n) HPR. We demonstrate here that the stimulating activity is the Werner syndrome protein (WRN). Although WRN contains both a 3'→5' helicase activity and a 3'→5' exonuclease activity, the stimulating activity was found to be the helicase activity, as a WRN helicase mutant failed to enhance (CTG)(n) HPR. Consistently, WRN efficiently unwound large (CTG)(n) hairpins and promoted DNA polymerase δ-catalyzed DNA synthesis using a (CTG)(n) hairpin as a template. We, therefore, conclude that WRN stimulates (CTG)(n) HPR on the template DNA strand by resolving the hairpin so that it can be efficiently used as a template for repair or replicative synthesis.

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Figures

FIGURE 1.
FIGURE 1.
DNA substrate and hairpin repair assay. A nicked circular DNA substrate containing a (CTG)25 hairpin in the continuous strand was constructed using M13mp18 bacterial phage as described (11). The blue and red lines represent CAG and CTG repeats, respectively, which are located between HindIII and EcoRI restriction enzyme sites of the phage DNA. The nick (at the BglI recognition position) is 164-bp 5′ to the hairpin. The green bar represents an oligonucleotide probe that anneals to the nicked strand near the BsrBI site. A schematic diagram of the CTG hairpin removal is shown, which is initiated by nick-directed incision opposite the hairpin, followed by hairpin unwinding and DNA resynthesis using the continuous strand as a template (11, 12). The repair product, which is 75 nt larger than the substrate, is detected by Southern hybridization using an oligonucleotide probe (i.e. the green bar) specifically annealing to the nicked strand.
FIGURE 2.
FIGURE 2.
Identification of WRN as a stimulating factor for CTG hairpin repair. A, HeLa nuclear activity, partially purified from a phosphocellulose P-11 column, stimulates CTG hairpin repair in reactions containing 50 μg of HeLa nuclear extract. B, protein and repair profiles of the phosphocellulose P-11 column are shown. HeLa nuclear extract was fractionated in a phosphocellulose P-11 column as described (28, 29). Samples (3 μl) of each fraction were tested for their ability to stimulate HPR in 50 μg of HeLa nuclear extract. The percentage of repair stimulation was calculated by subtracting the repair percentage from those of individual reactions supplemented with the P-11 fractions. Red curve shows repair stimulation (%). In each case, the stimulation was determined by subtracting the background repair (i.e. repair in reaction 1 in panel A) from the total repair. Black curve indicates protein concentration (mg/ml). C, Western blotting analysis shows the distribution of WRN, pol δ, and pol ϵ in the P-11 fractions used in panel A. D and E, purified recombinant WRN protein stimulates CTG hairpin repair.
FIGURE 3.
FIGURE 3.
WRN enhances pol δ-catalyzed DNA synthesis. A, diagram of the primer extension assay. The (CTG)35-containing circular M13mp18 ssDNA was linearized with restriction enzymes BstNI and BsrBI after annealing with oligonucleotides complementary to the corresponding restriction sequences. A 5′ 32P-labeled primer was then annealed with the M13mp18 template for DNA synthesis. B, pol δ-catalyzed primer extension using a (CTG)35 hairpin as a template in the presence of various amounts of purified WRN or RecQ1, as indicated. C, pol δ-catalyzed primer extension using a (CTG)35 hairpin as a template in the presence of wild-type WRN (W) or a helicase-deficient WRN mutant (M), as indicated. Reaction products were resolved in 6% denaturing polyacrylamide gels and visualized by a PhosphorImager. The total synthesis includes the product intermediates.
FIGURE 4.
FIGURE 4.
WRN resolves CTG hairpins. A, diagram of the unwinding-reannealing assay. 5′ 32P-labeled (CTG)15 or CTG)35 oligonucleotide was heated and slowly cooled down to ensure intra-strand hairpin formation. The hairpin substrate was incubated with purified WRN protein, followed by incubation with an unlabeled (CAG)15- or CAG)35-containing oligonucleotide, respectively, allowing for the formation of a dsDNA product with 5′ overhangs that is refractory to WRN unwinding and migrates differently from ssDNA in nondenaturing polyacrylamide gels. B, (CTG)35 hairpin unwinding by WRN or the K577M helicase mutant and its annealing with a (CAG)35 oligonucleotide in the presence or absence of ATP, as indicated. C, (CTG)15 hairpin unwinding by WRN or the K577M helicase mutant and its annealing with a (CAG)15 oligonucleotide in the presence of ATP. The unwinding-reannealing products were analyzed by nondenaturing polyacrylamide gel electrophoresis and detected by a PhosphorImager.
FIGURE 5.
FIGURE 5.
Proposed model for the role of WRN in stimulating CTG hairpin repair. pol δ is recruited to conduct repair DNA synthesis but is halted when encountering the CTG hairpin. By an interaction with pol δ, WRN is recruited to the site to resolve the CTG hairpin, which provides pol δ with an unstructured template, thereby stimulating CTG hairpin repair by enhancing repair DNA synthesis.
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