Switch-like control of helicase processivity by single-stranded DNA binding protein
- Department of Physics, University of Illinois, Urbana-Champaign, United States
- Center for Biophysics and Quantitative Biology, University of Illinois, Urbana-Champaign, United States
- Department of Biochemistry, Carver College of Medicine, University of Iowa, United States
- Center for the Physics of Living Cells, University of Illinois, Urbana-Champaign, United States
Figures
Figure 1 with 2 supplements
RPA2 increases xeroderma pigmentosum group D (XPD) helicase processivity.
(a) Schematic of FacXPD based on Sulfolobus acidocaldarius XPD structure (PDB 3CRV). XPD is composed of four domains: helicase domains 1 and 2 (HD1, pink; HD2, blue), which form the motor core, and …
Figure 1—figure supplement 1
DNA hairpin construct.
(a) The hairpin construct consists of three ligated DNA fragments: left (black) and right (blue) dsDNA handles and an 89 bp hairpin that includes a 5′ 10 nt ssDNA protein loading site. The left and …
Figure 1—figure supplement 2
Laminar flow chamber.
(a) Photograph of laminar flow chamber. Colored food dye highlights the different flow streams. Parafilm in the central channel is recolored to enhance contrast. The bead channels (yellow and green) …
Figure 2 with 1 supplement
Xeroderma pigmentosum group D (XPD) unwinds in bursts of varying processivity whose average increases with replication protein A 2 (RPA2) concentration.
(a) Representative trace of a single molecule of XPD unwinding in the presence of RPA2 (5 nM) at constant force (F = 12 pN). One XPD exhibits repetitive bursts of activity, making multiple attempts …
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Figure 2—source data 1
XPD burst processivity vs. [RPA2].
- https://cdn.elifesciences.org/articles/60515/elife-60515-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Xeroderma pigmentosum group D (XPD) burst processivity increases with replication protein A 2 (RPA2) concentration at a force of 9 pN.
Processivity of each burst (colored squares) for XPD alone (blue) and in the presence of RPA2 (10 nM; purple) at a constant force (F = 9 pN). The mean processivity (open squares) increases in the …
Figure 3 with 4 supplements
Replication protein A 2 (RPA2) transiently melts hairpin duplex but does not assist xeroderma pigmentosum group D (XPD) unwinding.
(a) Representative time trace of RPA2 transiently destabilizing hairpin dsDNA at a constant force (F = 12 pN). Inset: RPA2 melts ~8 bp, which then rapidly reanneals (see Figure 3—figure supplement 1)…
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Figure 3—source data 1
RPA2 melting statistics and XPD burst processivity vs. [gp32].
- https://cdn.elifesciences.org/articles/60515/elife-60515-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Replication protein A 2 (RPA2) transiently melts DNA under force.
(a) Representative time traces of RPA2 transiently destabilizing hairpin dsDNA at a constant force (F = 12 pN) and at varying RPA2 concentrations. The hairpin is exposed to varying concentrations of …
Figure 3—figure supplement 2
Detecting replication protein A 2 (RPA2) melting events during xeroderma pigmentosum group D (XPD) unwinding.
(a) Representative time traces of XPD unwinding in the presence of 2 and 50 nM RPA2 (data in gray) and corresponding step-fitting analysis (colored line) used to identify putative RPA2 melting …
Figure 3—figure supplement 3
Replication protein A 2 (RPA2) transient melting of DNA depends on force.
(a) Representative time traces of RPA2 transiently destabilizing hairpin dsDNA at a constant concentration (35 nM) and at different forces (F = 8–12 pN). The frequency of transient 5–10 bp dsDNA …
Figure 3—figure supplement 4
T4 gene protein 32 (gp32) transiently melts hairpin duplex but does not assist xeroderma pigmentosum group D (XPD) unwinding.
(a) Representative time trace of T4 gp32 transiently destabilizing hairpin dsDNA at a concentration of 250 nM and constant force of F = 8 pN. (b) Processivity of each burst (colored circles) for XPD …
Figure 4 with 2 supplements
Xeroderma pigmentosum group D (XPD) exhibits two burst types, the fraction of which is replication protein A 2 (RPA2) dependent.
(a) Plot of XPD unwinding bursts, aligned to start at t = 0 and grouped by RPA2 concentration (colored traces). XPD unwinding bursts come in two types: low processivity, never unwinding more than 25 …
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Figure 4—source data 1
High-processivity fraction and burst duration vs. [RPA2], and XPD speed vs. position.
- https://cdn.elifesciences.org/articles/60515/elife-60515-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Unwinding velocity varies with processivity type but not replication protein A 2 (RPA2) concentration.
(a, b) Average unwinding velocity as a function of hairpin position for low-processivity (a) and high-processivity (b) bursts at two representative RPA2 concentrations, 0 (blue) and 50 nM (green). …
Figure 4—figure supplement 2
Xeroderma pigmentosum group D (XPD) backward motion varies with processivity type but not replication protein A 2 (RPA2) concentration.
(a) Schematic representation of the sequestration model, in which RPA2 binding to ssDNA prevents XPD’s backward motion, enhancing unwinding. The model predicts that burst duration should increase …
Figure 5 with 2 supplements
A point mutation in xeroderma pigmentosum group D (XPD) enhances its processivity similarly to replication protein A 2 (RPA2).
(a) Representative traces of a single molecule of the mutant XPDH202A unwinding at constant force (F = 12 pN) alone (cyan) and in the presence of RPA2 (10 nM; magenta). (b) Processivity of …
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Figure 5—source data 1
XPD and mutant burst processivity vs. [RPA2], and complex formation statistics .
- https://cdn.elifesciences.org/articles/60515/elife-60515-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
A point mutation in xeroderma pigmentosum group D (XPD) enhances its processivity similarly to replication protein A 2 (RPA2) at a force of 9 pN.
(a) Processivity of individual bursts (colored squares) at a constant force of 9 pN for wild-type XPD alone (blue) and with 10 nM RPA2 (purple), and for XPDH202A alone (cyan) and with 10 nM RPA2 …
Figure 5—figure supplement 2
Test of stable complex formation.
(a) In the complex formation model, replication protein A 2 (RPA2) forms a complex with xeroderma pigmentosum group D (XPD) that activates it for processive unwinding. (b) Flow chamber configuration …
Figure 6
Replication protein A 2 (RPA2) activates a high-processivity state of xeroderma pigmentosum group D (XPD).
(a) Model of XPD enhancement by RPA2. Schematic of XPD–DNA complex (left; side and front views), with 5 nt of ssDNA (black) bound at a regulatory secondary binding site on HD1 that contains H202. …
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Figure 6—source data 1
High-processivity fraction vs. time and high-processivity state entry rate vs. [RPA2].
- https://cdn.elifesciences.org/articles/60515/elife-60515-fig6-data1-v2.xlsx
Tables
Table 1
Data statistics.
| Force (pN) | 7–8 | 9 | 12 | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| XPD | wt | wt | H202A | wt | H202A | ||||||||
| [RPA2] (nM) | 0 | 10 | 0 | 10 | 0 | 2 | 5 | 10 | 50 | 0 | 10 | ||
| [gp32] (nM) | 0 | 250 | |||||||||||
| No. of XPD molecules | 10 | 8 | 6 | 25 | 11 | 11 | 20 | 19 | 27 | 20 | 21 | 29 | 16 |
| No. of bursts | 141 | 23 | 54 | 74 | 38 | 67 | 94 | 123 | 79 | 60 | 82 | 123 | 79 |
| No. of bursts/XPD | 14.1 | 2.9 | 9 | 3 | 3.5 | 6.1 | 4.7 | 6.5 | 2.9 | 3.0 | 3.9 | 4.2 | 4.9 |
| No. of low-processivity bursts | 141 | 23 | 54 | 60 | 36 | 63 | 86 | 109 | 61 | 31 | 45 | 46 | 36 |
| No. of high-processivity bursts | 0 | 0 | 0 | 14 | 2 | 4 | 8 | 14 | 18 | 29 | 37 | 77 | 43 |
| XPD: xeroderma pigmentosum group D; RPA2: replication protein A 2; gp32: gene protein 32. | |||||||||||||
Table 2
Model fit parameters.
| Rate constant | Fit value (s−1) |
|---|---|
| k-1 | 0.18 ± 0.13 |
| k2 | 0.170 ± 0.017 |
| koff | 0.037 ± 0.019 |
| 0 | |
| k1, [RPA2]=0 nM | 0.02 ± 0.006 |
| k1, 2 nM | 0.059 ± 0.017 |
| k1, 5 nM | 0.065 ± 0.019 |
| k1, 10 nM | 0.200 ± 0.059 |
| k1, 50 nM | 1.15 ± 0.39 |
| Error bars represent 95% confidence intervals. RPA2: replication protein A 2. | |
