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. 2009;4(3):e4825.
doi: 10.1371/journal.pone.0004825. Epub 2009 Mar 13.

The Werner syndrome helicase/exonuclease processes mobile D-loops through branch migration and degradation

Patricia L Opresko  1 , Gregory Sowd, Hong Wang
Affiliations

"V体育官网入口" The Werner syndrome helicase/exonuclease processes mobile D-loops through branch migration and degradation

Patricia L Opresko (VSports app下载) et al. PLoS One. 2009.

Abstract

RecQ DNA helicases are critical for preserving genome integrity. Of the five RecQ family members identified in humans, only the Werner syndrome protein (WRN) possesses exonuclease activity VSports手机版. Loss of WRN causes the progeroid disorder Werner syndrome which is marked by cancer predisposition. Cellular evidence indicates that WRN disrupts potentially deleterious intermediates in homologous recombination (HR) that arise in genomic and telomeric regions during DNA replication and repair. Precisely how the WRN biochemical activities process these structures is unknown, especially since the DNA unwinding activity is poorly processive. We generated biologically relevant mobile D-loops which mimic the initial DNA strand invasion step in HR to investigate whether WRN biochemical activities can disrupt this joint molecule. We show that WRN helicase alone can promote branch migration through an 84 base pair duplex region to completely displace the invading strand from the D-loop. However, substrate processing is altered in the presence of the WRN exonuclease activity which degrades the invading strand both prior to and after release from the D-loop. Furthermore, telomeric D-loops are more refractory to disruption by WRN, which has implications for tighter regulation of D-loop processing at telomeres. Finally, we show that WRN can recognize and initiate branch migration from both the 5' and 3' ends of the invading strand in the D-loops. These findings led us to propose a novel model for WRN D-loop disruption. Our biochemical results offer an explanation for the cellular studies that indicate both WRN activities function in processing HR intermediates. .

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

VSports手机版 - Figures

Figure 1
Figure 1. Construction of mobile plasmid D-loops.
A. Schematic of 5′ Tail non-telomeric and telomeric (Tel) plasmid D-loops. The star denotes the 5′ end radiolabel. The invading strand of the 5′Tail Tel D-loop base pairs with the plasmid to form an 84 bp duplex with ten (TTAGGG) repeats flanked by 12 bp of unique sequence. The telomeric repeats are scrambled in the non-telomeric D-loop. Restriction enzyme sites are indicated as A, AvaI; B, BlpI. B. RecA generated plamid D-loops are stable. Plasmids (300 µM nucleotides) were mixed with the 5′end-labeled complementary oligonucleotide (3.6 µM nucleotides) and RecA protein (4 µM) (lanes 2 and 4). D-loops (Dlp) were then purified from unincorporated oligonucleotide (ss) (lanes 6 and 8). Reactions were run on a 4–20% polyacrylamide native gel. C. The D-loop invading strand is fully base paired with the plasmid strand. Reactions (20 µl) contained 50 pM purified 5′Tail non-telomeric (Non-Tel) or telomeric (Tel) D-loops and 10 units of the indicated restriction enzyme. Products were run on a 14% polyacrylamide denaturing gel.
Figure 2
Figure 2. WRN displaces and digests the invading strand of plasmid D-loops.
A. Reactions contained 50 pM of either the 5′Tail non-telomeric (lane 1–6) or the telomeric (Tel) (lanes 7–12) plasmid D-loops. The substrate was incubated with WRN concentrations increasing in doubling steps from 0.62 to 5 nM (lanes 2–5 or 8–11, respectively) for 15 min under standard reaction conditions and run on a 4–20% native polyacrylamide gel. ▴, heat denatured substrate. B. The 5′ Tail non-telomeric (lanes 1–8) or telomeric (lanes 9–15) were incubated with WRN concentrations increasing in doubling steps from 0.078 to 5 nM (lanes 2–8) and 0.15 to 5 nM (lanes 10–15) and run on a 14% denaturing polyacrylamide gel. Numbers represent oligonucleotide size markers. C. The percent total D-loop displacement from reactions in panel A were calculated as described in Materials and Methods and plotted against WRN concentration. WRN and 5′Tail plasmid D-loop, ▪ and dotted line; WRN and 5′Tail Tel plasmid D-loop, • and solid line. Values represent the mean and standard deviation (SD) from at least three independent experiments. D. WRN exonuclease is inactive on an 83 bp forked duplex. Reactions contained 0.25 nM of the 83-bp forked duplex containing ten telomeric repeats (thick black line) with 15 and 8 bp of flanking sequence. The substrate was incubated with WRN concentrations increasing in doubling steps from 0.19 to 25 nM for 15 min under standard reaction conditions. Reactions were run on a 14% denaturing gel.
Figure 3
Figure 3. WRN can branch migrate through the full 84 bp duplex to displace the invading strand.
Reactions contained 50 pM of either the 5′Tail non-telomeric (lanes 1–8) or the telomeric (Tel) (lanes 9–14 (A.) or 9–16 (B.)) plasmid D-loops. Reactions were run on 4–20% native polyacrylamide gels and visualized by phosphorimager analysis. ▴, heat denatured substrate. A. Branch migration after exonuclease inactivation with a WRN mutant. The substrate was incubated with E84A-WRN concentrations increasing in doubling steps from 0.15 to 5 nM (lanes 2–7) or from 0.62 to 5 nM (lanes 11–14) for 15 min under standard conditions. B. Branch migration after exonuclease inhibition with limiting Mg2+. The substrate was incubated with wild type WRN concentrations increasing in doubling steps from 0.15 to 5 nM (lanes 2–7, and lanes 10–15) for 15 min in reactions buffer containing 1 mM MgCl2 and 2 mM ATP. The percent total displacement was plotted against E84A-WRN (A.) or wild type WRN (B.) concentration. E84A-WRN or WRN and 5′Tail D-loop, ▪ and dotted line; E84A-WRN or WRN and 5′Tail Tel D-loop, • and solid line. Values are the mean and standard deviation (SD) from two-three independent experiments.
Figure 4
Figure 4. WRN exonuclease digests the long invading strands both prior to and after release from the plasmid D-loops.
A. Reactions contained 50 pM of either the 5′Tail non-telomeric (lane 1–8) or the telomeric (Tel) (lanes 9–15) plasmid D-loops. The substrate was incubated with WRN concentrations increasing by doubling steps from 0.15 to 5 nM (lanes 2–7) or from 0.31 to 5 nM (lanes 10–14) for 15 min under standard reaction conditions except ATP was replaced with ATPγS. Reactions were run on 4–20% native polyacrylamide gels and visualized by phosphorimager analysis. ▴, heat denatured substrate. B. The percent total displacement was calculated as described in Materials and Methods and plotted against WRN concentration. WRN and 5′Tail D-loop, ▪ and dotted line; WRN and 5′Tail Tel D-loop, • and solid line. Values are the mean and standard deviation (SD) from two independent experiments. C. and D. WRN exonuclease on intact and released invading strands. Reactions contained 50 pM of either the non-telomeric (C.) 5′Tail D-loop (lanes 1–7) or 120-mer oligonucleotide (lanes 11–14) or the telomeric (D.) 5′Tail Tel D-loop (lanes 7–12) or 120-mer oligonucleotide (lanes 1–4). The substrates were incubated with WRN (5 nM) or E84A-WRN mutant (X; 5 and 2.5 nM) under standard conditions for 15 min except that either 2 mM ATP, 2 mM ATPγS (γ) or no ATP (-) was added as indicated. Reactions were run on 14% polyacrylamide denaturing gels. Oligonucleotide size markers were loaded for reference.
Figure 5
Figure 5. Structural requirements for WRN activity on plasmid D-loops.
A. Schematic of 3′Tail and No Tail D-loops. The star denotes the 5′ end radiolabel. B. Reactions contained 50 pM of either the 3′Tail (lane 1–6) or the No Tail (lanes 7–12) plasmid D-loops. The substrate was incubated with WRN concentrations increasing by doubling steps from 0.62 to 5 nM (lanes 2–5 and 8–11) for 15 min under standard reaction conditions. Reactions were run on 4–20% polyacrylamide native gels and visualized by phosphorimager analysis. ▴, heat denatured substrate. C. The percent total displacement was calculated as described in Materials and Methods and plotted against WRN concentration. WRN and 3′Tail D-loop, ▪ and dotted line; WRN and No Tail D-loop, • and solid line. Values are the mean and standard deviation (SD) from at least three independent experiments. D. Analysis of WRN exonuclease activity. Reactions contained 50 pM of either the 3′Tail (lanes 1–4 and 9–10) or the No Tail (lanes 5–8 and 11–12) D-loops. The substrates were incubated with 5 nM WRN or E84A-WRN mutant (X) under standard conditions for 15 min except that either 2 mM ATP, 2 mM ATPγS (γ) or no ATP (-) was added as indicated. Reactions were run on 14% polyacrylamide denaturing gels. Arrow points to products representing digestion almost to the end of the 84-bp duplex.
Figure 6
Figure 6. Kinetics of WRN activity on plasmid D-loops.
Reactions contained 50 pM of either the 5′Tail, No Tail, or 3′Tail plasmid D-loops as indicated. The 5′ labeled end is indicated with a star. The substrate was incubated with 5 nM WRN (Panel A–B) or 5 nM E84A-WRN mutant (Panel C) for 0.5, 1, 2, 4, 8, 15, 30 min under standard reaction conditions. Reactions were run on 4–20% polyacrylamide native gels and visualized by phosphorimager analysis. ▴, heat denatured substrate. B. and C. The percent total displacement was calculated as described in Materials and Methods and plotted against time for reactions with wild type WRN (B.) or the E84A-WRN mutant (C.). 5′Tail D-loop, ▪ and black line; 3′Tail D-loop, ▴ and blue line; No Tail D-loop, ▾and yellow line. The representative phosphorimage scans for the E84A-WRN reactions are shown in Supplemental Fig. S3A.
Figure 7
Figure 7. WRN recognized both ends of the invading strand duplex in a plasmid D-loop.
A schematic is shown of the plasmid D-loops with a invading strand that forms a 60 bp duplex containing 18 nt protruding 5′ and 3′ ssDNA tails. A biotinylated nucleotide (circle) was positioned 2 nt from the duplex on the 3′ and 5′ tails (A), the 5′tail only (B) or the 3′tail only (C). Reactions contained 50 pM of plasmid D-loop that was pre-incubated for 10 min in reaction buffer alone or together with streptavidin as indicated. Then WRN (1.25 nM) was added and reaction aliquots were terminated at 0, 1, 3, 5, 10, 15 and 30 min. Reactions were run on 4–20% native polyacrylamide gels and visualized by phosphorimager analysis. ▴, heat denatured substrate. Dlp, D-loop; sb/ss, streptavidin-bound biotin ssDNA; b/ss biotin ssDNA. The percent total displacement was calculated as described in Materials and Methods and plotted against time. Biotin containing D-loops, ▪ and dotted line; Biotin+Strepavidin containing D-loops, • and solid line. Values represent the mean and standard deviation (SD) from at least three independent experiments. D. WRN exonuclease activity on ssDNA is inhibited by a biotin moiety. Reactions contained 50 pM of the 96-mer 5B Tail (lanes 1–7) or 3B Tail (lanes 8–14) oligonucleotides (Table 1). The substrates were incubated with 1.25 nM WRN and terminated after 0, 1, 3 5, 10, 15 and 30 reaction time under standard conditions. Reactions were run on 14% polyacrylamide denaturing gels.
Figure 8
Figure 8. WRN disruption of D-loops with a 5′ blocked junction does not require a 3′tail or exonuclease activity.
A schematic is shown of the plasmid D-loop with an invading strand that forms a 60 bp duplex containing a single 18 nt protruding 5′ ssDNA tail. A biotinylated nucleotide (circle) was positioned 2 nt from the duplex on the 5′ tail. Reactions contained 50 pM of plasmid D-loop that was pre-incubated for 10 min in reaction buffer alone or together with streptavidin as indicated. Then WRN (1.25 nM) was added and reaction aliquots were terminated at 0, 1, 3, 5, 10, 15 and 30 min. Reactions were run on 4–20% native polyacrylamide gels and visualized by phosphorimager analysis. ▴, heat denatured substrate. Dlp, D-loop; sb/ss, streptavidin-bound biotin ssDNA; b/ss biotin ssDNA. The percent total displacement was calculated as described in Materials and Methods and plotted against time. Biotin containing D-loops, ▪ and dotted line; Biotin+Strepavidin containing D-loops, • and solid line. Values represent the mean and standard deviation (SD) from two independent experiments.
Figure 9
Figure 9. Bidirectional model for WRN branch migration and exonuclease activity on mobile D-loops.
WRN protein with the helicase (triangle) and exonuclease (crescent) domains are depicted as separate monomers, but could also be linked in a higher oligomeric complex (denoted by a dotted line) such as a tetramer. The D-loops represent an intermediate in recombination, SDSA, and a structure at the telomeric end or ALT pathway. The D-loop depicted contains no pre-existing ssDNA tail although such tails may occur in vivo, such as a 3′ tail in a non-productive D-loop. WRN protein recognizes the 5′ duplex junction and helicase translocation along the plasmid strand in a 3′ to 5′ direction will promote branch migration. WRN also recognizes the 3′ duplex junction and may translocate along the invading strand 3′ to 5′ to promote branch migration, but can also digest the strand 3′ to 5′. In this model helicase driven branch migration proceeds in a bidirectional manner toward the middle causing shrinking of the displacement loop and subsequent release of the invading strand. Branch migration progresses more rapidly than the exonuclease which digests at one end and may cause a “loop out” of ssDNA (last structure). WRN exonuclease activity can also digest the strand after release depending on the length (not depicted here).
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VSports注册入口 - References

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