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. 2023 Jan 11;51(1):337-348.
doi: 10.1093/nar/gkac1200.

Werner syndrome protein works as a dimer for unwinding and replication fork regression

Soochul Shin  1 , Kwangbeom Hyun  2 , Jinwoo Lee  1 , Dongwon Joo  1 , Tomasz Kulikowicz  3 , Vilhelm A Bohr  3 , Jaehoon Kim  2 , Sungchul Hohng  1
Affiliations

Werner syndrome protein works as a dimer for unwinding and replication fork regression

Soochul Shin et al. Nucleic Acids Res. .

Abstract

The determination of the oligomeric state of functional enzymes is essential for the mechanistic understanding of their catalytic activities. RecQ helicases have diverse biochemical activities, but it is still unclear how their activities are related to their oligomeric states. We use single-molecule multi-color fluorescence imaging to determine the oligomeric states of Werner syndrome protein (WRN) during its unwinding and replication fork regression activities. We reveal that WRN binds to a forked DNA as a dimer, and unwinds it without any change of its oligomeric state. In contrast, WRN binds to a replication fork as a tetramer, and is dimerized during activation of replication fork regression. By selectively inhibiting the helicase activity of WRN on specific strands, we reveal how the active dimers of WRN distinctly use the energy of ATP hydrolysis for repetitive unwinding and replication fork regression.

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Figures

Figure 1.
Figure 1.
Oligomeric states of WRN that differentially bind to a forked DNA and a replication fork. (A) A design of a forked DNA. (B) A design of a model replication fork. Orange lines represent heterologous bases. (C) The experimental procedure to assess the oligomeric states of WRN. (D) Representative fluorescence intensity time traces of Cy3 (green), Cy5 (red) and GFP (blue). After Cy3 and Cy5 imaging for co-localization, GFP-tagged WRN was injected at t = 0. The first dashed line indicates the timing of buffer exchange from the Cy3/Cy5 imaging buffer with oxygen scavenging system to the GFP imaging buffer without oxygen scavenging system. The second dashed line (black arrow) indicates the injection of GFP-tagged WRN. Red arrow indicates the photo-bleaching steps of GFP. (E) Distribution of photobleaching steps for forked DNA, and model replication fork.
Figure 2.
Figure 2.
Unwinding of forked DNA by WRN dimer (A) The experimental scheme to check the unwinding activity of WRN after observing the oligomeric state of WRN binding to the forked DNA. (B) Representative fluorescence intensity time traces of the experiment (A). Photobleaching steps of GFP were counted in the absence of ATP. After the ATP injection, repetitive unwinding of the forked DNA by WRN was observed. (C) Distribution of photobleaching steps of experiment (B). (D) The experimental scheme to observe the oligomeric state of WRN that are repetitively unwinding the forked DNA. (E) Representative fluorescence intensity time traces of the experiment. Photobleaching steps of GFP were counted in the presence of ATP, and the unwinding activity of WRN was checked using FRET in the Cy3/Cy5 imaging buffer. (F) Distribution of photobleaching steps of experiment (E).
Figure 3.
Figure 3.
Tetramer-to-dimer transition of WRN for replication fork regression. (A) The experimental scheme to check the unwinding activity of WRN after observing the oligomeric state of WRN binding to the model replication fork. (B) Representative fluorescence intensity time traces of the experiment (A). Photobleaching steps of GFP were counted in the absence of ATP. After the ATP injection, replication fork regression was observed. (C) Distribution of photobleaching steps of experiment (B). (D) The experimental scheme to observe the oligomeric state of WRN that is doing repetitive replication fork regression. (E) Representative fluorescence intensity time traces of the experiment (D). Photobleaching steps of GFP were counted in the presence of ATP, and the replication fork regression activity of WRN was checked using FRET in the Cy3/Cy5 imaging buffer. (F) Distribution of photobleaching steps of experiment (E).
Figure 4.
Figure 4.
Unwinding activity of WRN on DNA/RNA hybrid substrates. (A) DNA/RNA hybrid sample with a 3′-overhang RNA strand (left). The RNA region is indicated by cyan. Representative fluorescence intensity time traces of Cy3 (green) and Cy5 (red) in the presence of 1 mM ATP and 1nM RPA (right). No molecule (0/698) shows unwinding behavior. 26 nM wild type WRN was used. (B) DNA/RNA hybrid sample with a 5′-overhang RNA strand (left). Representative intensity time traces of Cy3 (green) and Cy5 (red) in the presence of 1 mM ATP and 1nM RPA (right). 30.9% (224/725) of molecules show unwinding behavior. 26 nM wild type WRN was used.
Figure 5.
Figure 5.
Replication fork regression activity of WRN on DNA/RNA hybrid substrates. (A–G, left) Schematics of sample designs. The RNA, and heterologous regions are indicated by cyan, and orange, respectively. (A–G, right) Representative fluorescence intensity time traces of Cy3 (green) and Cy5 (red) (right). Dashed lines indicate the 1 mM ATP injection timing for the initiation of replication fork regression. In (E) and (F), experiments were performed in the presence of 1 mM ATP. 26 nM wild type WRN was used in all experiments. The number of molecules showing activities are (A) (0/780), (B) (190/625), (C) (250/732), (D) (0/712), (E) (175/633), (F) (158/615) and (G) (208/705).
Figure 6.
Figure 6.
Mechanistic models of repetitive unwinding, and replication fork regression. (A) In solution, both dimeric and tetrameric forms of WRN coexist. Dimeric WRN preferentially binds to the forked DNA, and unwinds it with one protomer translocating on the 3′-overhang strand and the other anchoring on the 5′-overhang strand. As the unwinding progresses, mechanical stress accumulates, and rewinding abruptly occurs via the sliding back mechanism once a threshold is passed. (B) Tetrameric WRN preferentially binds to a stalled replication fork, and is converted into a dimer for the activation of the replication fork regression. During branch migration, the two protomers of the dimeric WRN translocate on either the parental lagging strand or the daughter leading strand.
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