Amidst multiple binding orientations on fork DNA, Saccharolobus MCM helicase proceeds N-first for unwinding
- Baylor University, United States
Figures
Figure 1 with 4 supplements
SsoMCM orientation mapping onto equal arm fork DNA substrates.
(A) APB or (B) FeBABE orientation mapping of the 3’- encircled strand labelled at the 5’- duplex end with Cy3 on an equal arm fork DNA substrate with a 20 base duplex (DNA164/165-3). SsoMCM was …
Figure 1—figure supplement 1
Crosslinking MCM mutants to DNA.
The UV crosslinked protein-DNA product of two SsoMCM mutants and the uncrosslinked SsoMCM C642A were run on a 10% SDS- PAGE gel. The shifted protein-DNA product is indicated by a black arrow (←). In …
Figure 1—figure supplement 2
Validation of MCM-APB cleavage on ssDNA.
(A) APB cleavage of a 5’-Cy3 labelled 50 nt ssDNA. No cutting in the absence of APB or MCM (lanes 2–4). MCM-APB can cleave DNA in the absence or presence of UV or NaOH (lanes 5, 8–11). (B) APB …
Figure 1—figure supplement 3
DNA binding by fluorescence anisotropy.
DNA binding was determined by fluorescence anisotropy for each of the DNA substrates. The binding constants (Kd) for equal arm fork DNA substrates (5’-Cy3 labelled or 3’-Cy3 labelled), 3’- long arm …
Figure 1—figure supplement 4
DNaseI footprinting and EMSA.
(A) DNaseI digestion of fork DNA in the absence (lane 3) and presence of SsoMCM (lanes 4–5) at different ratios. Fork DNA is labelled at the 3’ position on the duplex end with Cy5. Markers at 20 nt …
Figure 2
SsoMCM orientation mapping onto 3’-(DNA171/165-3) or 5’-(DNA172/164-5) long arm fork DNA substrates.
(A) APB orientation mapping of the 3’-encircled strand labelled at the 5’ duplex end with Cy3 on a 3’-long arm fork DNA substrate with a 20 base duplex. SsoMCM was labelled with APB at C682 (within …
Figure 3
SsoMCM (-WH) orientation mapping onto fork DNA substrates.
(A) Schematic of full length SsoMCM highlighting the NTD (orange), CTD (blue), and the WH domain (grey) and cysteine sites of conjugation used. (B) Orientation mapping of SsoMCM labelled at C452 or …
Figure 4 with 1 supplement
Single turnover DNA unwinding.
(A) DNA unwinding of equal arm (in blue boxes -■-, 5’-Cy3 labelled at the duplex), 3’-long arm (in green lower left triangle -◣-, 5’-Cy3 labelled at the duplex), 5’-long arm (in red lower right …
Figure 4—figure supplement 1
Single turnover DNA Unwinding.
DNA unwinding of 3’-long arm/5’-arm (n), (n = 0, in green open lower left triangle -◣-, n = 8 in green closed lower left triangle -◣-,5’-Cy3 labelled at the duplex), 5’-long arm/3’-arm (n), (n = 0, …
Figure 5
Steady-state FRET quenching of SsoMCM bound to a fork DNA.
(A) Schematic of the DNA fork substrate that includes a 30 nt 3’-arm, 20 nt 5’-arm, a biotin placed nine nt from the duplex junction and a dT-FAM placed a further six nt downstream. The Tm of the …
Figure 6 with 1 supplement
Presteady-state stopped-flow measure of SsoMCM translocation/unwinding.
SsoMCM (167 nM hexamer) labelled at (A, C) C682 or (B, D) the N-terminus was preassembled onto 3’-long arm fork DNA (125 nM) with a 30 base 3’-arm and a 7 base 5’-arm in the (A, B) presence or (C, D)…
Figure 6—figure supplement 1
Presteady-state loading of SsoMCM on DNA.
https://doi.org/10.7554/eLife.46096.013
Figure 7
Presteady-state stopped-flow measure of SsoMCM translocation.
SsoMCM (120 nM hexamer) labelled at C682 (orange) or the N-terminus (blue) was preassembled onto (A) 3’- or (B) 5’long arm ssDNA (100 nM) with a flanking 20 bp duplex with 375 nM streptavidin. A FAM …
Figure 8
SsoMCM loading at origins.
Model for loading double hexamer MCM at an origin of replication and the two pathways (i or ii) for encircling the 5’-3’ or 3’-5’ strands placing the CTD at the duplex (C@duplex). Translocation from …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Plasmid construct (E. coli) | pET30a-SsoMCM (C642A) | McGeoch et al. (2005) | ||
| Plasmid construct (E. coli) | pET30a-SsoMCM (C682A) | McGeoch et al. (2005) | ||
| Plasmid construct (E. coli) | pET30a-SsoMCM 1–612 (G452C) | This paper | Site- directed mutagenesis using primers listed in Materials and methods | |
| Plasmid construct (E. coli) | pET30a-SsoMCM1–612 (S456C) | This paper | Site- directed mutagenesis using primers listed in Materials and methods | |
| Expression strain | Rosetta 2 | Novagen | ||
| Chemical compound | 4-azidophenacyl bromide (APB) | Sigma-Aldrich | 57018-46-9 | |
| Chemical compound | ATP | Invitrogen | 51963-61-2 | |
| Chemical compound | 1-(p-Bromoacetamidobenzyl) ethylenediamine N, N,N (Fe-BABE) | Dojindo | 186136-50-5 | |
| Chemical compound | DNaseI | New England Biolabs | M0303S | |
| Chemical compound | Streptavidin | Invitrogen | 800-955-6288 | |
| Chemical compound | Cy3 succinimidyl ester | ThermoFisher | 57757-57-0 | |
| Chemical compound | Cy3 maleimide | ThermoFisher | 45-001-273 | |
| Sequence-based reagent | DNA primers and substrates | Sigma-Aldrich and IDT | Refer to Materials and methods | |
| Software, algorithm | Kaleidagraph | www.synergy.com | V4.5 |
Additional files
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Supplementary file 1
Table and listing of all DNA sequences and templates used.
- https://doi.org/10.7554/eLife.46096.016
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Transparent reporting form
- https://doi.org/10.7554/eLife.46096.017
