The pre-sensor 1 (PS1) hairpin is found in helicases of the AAA+ family (ATPases associated with a variety of cellular activities) of proteins and is implicated in DNA translocation during DNA unwinding. To determine whether the PS1 b hairpin is required in the eukaryotic replicative helicase, Mcm2-7 (also comprised of AAA+ proteins), we mutated the conserved lysine residue in the PS1 hairpin in each of the S. cerevisiae Mcm subunits to alanine. Only the PS1 hairpin of Mcm3 was essential for viability, while mutation of the PS1 hairpin in the remaining Mcm subunits resulted in minimal phenotypes, with the exception of Mcm7. The viable alleles were synthetic lethal with each other. Mcm2-7 containing Mcm3K499A (Mcm2-73K499A) disrupts helicase activity, yet the ATPase activity of Mcm2-73K499A was similar to the wild type Mcm2-7, and its interaction with single-stranded DNA was subtly altered in vitro. These findings indicate that the PS1 hairpins in the Mcm2-7 subunits have important and distinct functions most evident with Mcm3K499A
