Having observed stimulation of CMG by Mcm10 in helicase assays, we wished to determine whether Mcm10 also stimulated CMG in the context of the DNA replication fork. To do so, we used a 2.8 kb linear forked DNA primed with a 32P-5’ end-labeled 37mer oligo (Georgescu et al., 2014; Langston et al., 2014) and tested the effect of Mcm10 on extension of the radiolabeled primer by the core leading strand replisome consisting of CMG, Pol ε, RFC, PCNA, and RPA (Figure 4). CMG is pre-incubated with the DNA substrate (in the presence or absence of Mcm10) for 5’ at 30° C to allow CMG loading onto the 3’ tail of the fork (see reaction scheme in Figure 4A). ATP is omitted at this stage to prevent CMG from unwinding the DNA before assembly of the replisome. Pol ε, RFC, PCNA and 2 dNTPs are then added and incubated a further 4’ to assemble the core leading strand replisome and the reaction is started upon addition of the remaining dNTPs and ATP along with RPA. Analysis of the autoradiogram in Figure 4B shows a modest enhancement of CMG-dependent primer extension by Mcm10. In the presence of Mcm10 the 2.8 kb full length product can be observed at the 6‘ time point (Figure 4B, lanes 8 and 13), yielding a maximum fork rate of 7.8 nucleotides (ntds)/s; 467 ntds/min), while 8’ is required to observe the full-length product in the absence of Mcm10 (Figure 4B, lane 4; 5.8 ntds/s; 350 ntds/min). This result is consistent with modest stimulation of the rate of DNA synthesis by Mcm10 in an origin-dependent plasmid replisome assembly assay (Lõoke et al., 2017).