Figure 3. CMG+Mcm10 strand interactions during duplex translocation.

(A) Diagram of the naturally occurring negatively charged phosphodiester linkage in the phosphate backbone of a DNA chain (left) and the uncharged methylphosphonate linkages used in these experiments (right). (B) The experiment from Figure 2C (CMG+Mcm10) was repeated using a T30 substrate with 10 neutral methylphosphonate linkages (shown in pink in the schematics above the gel) in the 5’-3’ strand (left) or in the 3’-5’ strand (right) of the duplex. See Figure 3—figure supplement 2 and Supplementary file 1 for further details on the substrate. The * next to the gels indicates gel-shift of the substrate by CMG+Mcm10. (C) A plot of the data from (B) shows the averages of three independent trials using these substrates. The error bars show the standard deviations. Also see Figure 3—figure supplement 1.

Figure 3—figure supplement 1. Duplex strand ATPases contact only one strand while encircling duplex DNA.

(A) The E. coli clamp loader binds only one strand during action on dsDNA. (B) CMG motor domains bind ssDNA in a B-form conformation. (C) Little to no conformation change would be needed for CMG motors to bind B-form ssDNA and then slide onto dsDNA (flush end) and continue tracking on the same 3’−5’ strand.

© 2009 Elsevier

Panel A is reprinted from Simonetta et al., 2009, with permission from Elsevier. It is not covered by the CC-BY 4.0 licence and further reproduction of this panel would need permission from the copyright holder.

© 2018 Springer Nature

Panel B is reprinted from O'Donnell and Li, 2018, with permission from Springer Nature. It is not covered by the CC-BY 4.0 licence and further reproduction of this panel would need permission from the copyright holder and further reproduction of this panel would need permission from the copyright holder.

Figure 3—figure supplement 2. Design of substrates with methylphosphonate linkages in Figure 3.

To investigate how the CMG+Mcm10 motor interacts with the duplex DNA in its central channel, we used derivatives of oligos T30A or T30B (sequences in Supplementary file 1) in which a stretch of ten bases (shown in green in the DNA sequence) is connected by neutral methylphosphonate linkages (chemical schematic at top) instead of the normal negatively charged phosphodiester linkages on one strand or the other. The methylphosphonate derivate oligos are designated “T30A MP” and “T30B MP” (Supplementary file 1).