To test the hypothesis that Mcm10 may help CMG overcome blocks, we examined the effect of Mcm10 on CMG unwinding of forked DNA with strand-specific blocks on either the lagging or leading strand of the duplex portion of the DNA (Figure 5 and Figure 5—figure supplement 1, respectively). In these experiments, the block is formed by two biotinylated nucleotides on one DNA strand or the other, spaced 6 bp apart, that are tightly bound by the 53 kDa streptavidin protein. We recently showed that CMG unwinding is inhibited by biotin-streptavidin on either strand and is nearly inactive when two biotin-streptavidin blocks are present on either strand of the duplex (Langston and O'Donnell, 2017). This is unique to CMG, as thus far all other hexameric helicases examined are not blocked by an obstruction on the non-tracking strand and are only blocked by an obstruction on the translocating strand (Hacker and Johnson, 1997; Kaplan, 2000; Kaplan et al., 2003; Lee et al., 2014; Nakano et al., 2013). Inhibition of hexameric helicases by a block on one strand but not the other is interpreted as a steric exclusion mode of unwinding in which the non-tracking strand is completely excluded from the central channel of the helicase ring, thereby explaining why bulky substituents on the non-tracking strand do not affect helicase unwinding.