Mcm10 promotes rapid isomerization of CMG-DNA for replisome bypass of lagging strand DNA blocks
- The Rockefeller University, United States
- Howard Hughes Medical Institute, United States
Figures
Figure 1
CMG-fork structure and isomerization needed for bypass of lagging strand blocks.
(A) Illustration of a hexameric helicase in the steric exclusion mode, encircling the tracking strand and excluding the non-tracking strand from the central channel. (B) CMG-forked DNA structure …
Figure 2 with 5 supplements
Mcm10 binds CMG and stimulates its helicase activity.
(A) Titration of Mcm10 in a CMG unwinding assay. Reactions contained 25 nM CMG with either: no Mcm10 (lanes 2–4), 25 nM Mcm10 (1:1, lanes 5–7), 50 nM Mcm10 (2:1, lanes 8–10) or 100 nM Mcm10 (4:1, …
Figure 2—figure supplement 1
Mcm10 does not have helicase activity.
Helicase assays were performed as in Figure 2 using Mcm10 only (lanes 3–5), CMG only (lanes 6–8) or CMG + Mcm10 (lanes 9–11).
Figure 2—figure supplement 2
Neither yeast RPA nor E. coli SSB stimulates CMG helicase.
As shown in the schematic at the top, CMG (20 nM) was pre-incubated with the 160-mer duplex substrate from Figure 3B (0.5 nM) for 10’ followed by addition of ATP (1 mM) to start the CMG unwinding …
Figure 2—figure supplement 3
SDS PAGE of flag bead eluted CMG-Mcm10.
To determine the stoichiometry of GINS in the CMG-Mcm10 complex, a Flag purification of CMG-Mcm10 was analyzed in a 10% PAGE gel followed by densitometric analysis. The result indicates the …
Figure 2—figure supplement 4
MonoQ reconstitution of CMG/Mcm10 complex.
To form a CMG-Mcm10 complex, 600 μg purified CMG (765 pmol) was mixed with 250 μg purified Mcm10 (3.1 nmol). The mixture was incubated on ice for 30’ and spun in a microcentrifuge at 15,000 rpm for …
Figure 2—figure supplement 5
The CMG-Mcm10 complex reconstituted on MonoQ is functional.
30 nM (final concentration) of reconstituted CMG-Mcm10 complex (Figure 2—figure supplement 4) was preincubated with the 50 bp duplex fork substrate (0.5 nM) for 10’ in the absence of ATP in buffer …
Figure 3 with 1 supplement
Mcm10 enhances the processivity of CMG unwinding.
(A) Scheme of the reaction. CMG was pre-incubated with the substrates for 10’ before addition of ATP ± a 2-fold excess of Mcm10 over CMG. (B) Native PAGE analysis of CMG unwinding in the absence …
Figure 3—figure supplement 1
Delayed replication of longer duplex allows estimation of unwinding rate.
A helicase assay identical to those in Figure 3 was performed except that both the 50 bp duplex and 160 bp duplex for substrates were added to the reaction at 0.5 nM each substrate and pre-incubated …
Figure 4
Mcm10 stimulates CMG-dependent leading strand replication in the absence of Mrc1-Tof1-Csm3.
(A) Reaction scheme. See Materials and methods for details. (B) Alkaline agarose gel of leading strand products synthesized by the core leading strand replisome with the indicated amounts of Mcm10. …
Figure 5 with 1 supplement
Mcm10, but not MTC complex, enables CMG and the replisome to bypass a lagging strand block.
(A) Illustration of the helicase reaction scheme. See Materials and methods for details. (B) Unwinding assays on dual biotinylated forks with or without streptavidin and with or without Mcm10, as …
Figure 5—figure supplement 1
A leading strand block inhibits CMG unwinding even in the presence of Mcm10.
Helicase reactions were performed as in Figure 5B except that the substrate contained a dual biotin-streptavidin block on the leading strand (50duplex LEAD2 dual biotin); the lagging strand …
Figure 6 with 1 supplement
Lagging strand blocks are not displaced from DNA during unwinding by CMG-Mcm10.
(A) The helicase reaction in Figure 5B (CMG + Mcm10) was repeated with the radiolabel on the biotinylated lagging strand and in the presence of free biotin as a trap for displaced streptavidin (See …
Figure 6—figure supplement 1
Free biotin prevents streptavidin binding to the biotinylated oligos but does not displace pre-bound streptavidin.
To test the effectiveness of the free biotin trap used in Figure 6, the radiolabeled, dual-biotinylated oligos (0.5 nM) were incubated with streptavidin (4 μg/ml) and biotin (1.5 μM) was added …
Figure 7
Model of Mcm10 function.
Proposed model of Mcm10 mediated isomerization of CMG-DNA at a replication fork. Left, CMG encounters an impediment on the DNA but cannot pass it because it surrounds dsDNA in the N-terminal tier of …
Tables
Table 1
Oligonucleotides Used in this Study.
All oligonucleotides used in this study were ordered from IDT with the indicated modifications.
| Oligo name | Sequence (5’ to 3’) | Modification(s) |
|---|---|---|
| 50dupex LAG | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACGCTGCCGAATTCTGGCTTGCTAGGACATTACAGGATCGTTCGGTCTC | None |
| 50duplex LAG dual biotin | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACGCTGCCGAATTCTGGCTTGCTAGGACATTACAGGATCGTTCGGTCTC | Two biotin-modified thymidine residues in BOLD |
| 50duplex LEAD | GAGACCGAACGATCCTGTAATGTCCTAGCAAGCCAGAATTCGGCAGCGTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT*T*T*T*T*T*T | The six dT residues at the 3’ end are connected by phosphorothioate bonds (*) |
| 50duplex LAG2 | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACGCTGCCGAATTCTGGATTGCTAGGACATTACAGGATCGTTCGGTCTC | None |
| 50duplex LEAD2 dual biotin | GAGACCGAACGATCCTGTAATGTCCTAGCAATCCAGAATTCGGCAGCGTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT*T*T*T*T*T*T | Two biotin-modified thymidine residues in BOLD; the six dT residues at the 3’ end are connected by phosphorothioate bonds (*) |
| 160mer duplex LEAD | AGAGAGTAGAGTTGAGTTGTGATGTGTAGAGTTGTTGTAGAGAAGAGTTGTGAAGTGTTGAGTAGAGAAGAGAAGAGAAGTGTTGTGATGTGTTGAGTAGTGTAGAGTTGAGAAGTAGAGATGTGTTGAGATGAGAAGAGTTGTAGTTGAGTTGAAGTGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT*T*T*T*T*T | The five dT residues at the 3’ end are connected by phosphorothioate bonds (*) |
| 160mer duplex LAG | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCCACTTCAACTCAACTACAACTCTTCTCATCTCAACACATCTCTACTTCTCAACTCTACACTACTCAACACATCACAACACTTCTCTTCTCTTCTCTACTCAACACTTCACAACTCTTCTCTACAACAACTCTACACATCACAACTCAACTCTACTCTCT | None |
| Blocked Fork LEAD | ACCGGAGACCGAACGATCCTGTAATGTCCTAGCAAGCCAGAATTCGGCAGCGTCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGGAAAGAATGTTGGTGAGGGTTGGGAAGTGGAAGGATGGGCTCGAGAGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTT*T*T*T*T*T | The five dT residues at the 3’ end are connected by phosphorothioate bonds (*) |
| Blocked Fork LAG | TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGACGC TGCCGAATTCTGGCTTGCTAGGACATTACAGGATCGTTCG*G*T*C*T*C | Two biotin-modified thymidine residues in BOLD; the five dT residues at the 3’ end are connected by phosphorothioate bonds (*) |
| Blocked Fork Primer | CCTCTCGAGCCCATCCTTCCACTTCCCAACCCTCACC | None |
| C2 | CCTCTCGAGCCCATCCTTCCACTTCCCAACCCTCACC | None |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.29118.018
