Mini-chromosome maintenance (MCM) proteins form complexes that are required for DNA replication and are highly conserved throughout evolution. The replicative helicase of eukaryotic organisms is composed of the six paralogs MCM2-7, which form a heterohexameric ring structure. In contrast, the structure of the archaean replicative MCM helicase is a single Mcm protein that forms a homohexameric complex. Atomic structures of archaeal MCMs have identified multiple beta-finger structures in Mcm proteins whose in vivo function is unknown. In the present study, we have investigated the physiological role of the pre-sensor 1 beta-hairpin (PS1-hp) beta-fingers of Saccharomyces cerevisiae Mcm4p and Mcm5p in DNA replication initiation and elongation in vivo. The PS1-hp beta-finger mutant of Mcm5p (mcm5-HAT K506A::URA3) has a growth defect at both 18° and 37°. Mutation of the Mcm4p PS1-hp beta-finger (mcm4-HA K658A::TRP1) does not have a growth defect, indicating different functional contributions of the PS1-hp beta-finger structures of different MCM helicase subunits. Both Mcm4p and Mcm5p PS1-hp beta-finger mutants can coimmunoprecipitate Mcm2p, indicating the formation of the hexameric MCM helicase complex. Both PS1-hp beta-finger mutants have a plasmid loss phenotype that is suppressible by origin dosage, indicating a defective replication initiation. Surprisingly, a defect in the binding of PS1-hp MCM mutants to origins of DNA replication was not found by chromatin immunoprecipitation, suggesting a novel interpretation in which the defect is in a subsequent step of DNA strand separation by the MCM helicase. The double mutant mcm4-HA K658A::TRP1 mcm5-HAT K506A::URA3 is lethal, displaying a terminal MCM mutant phenotype of large budded cells.