The role of ATP hydrolysis by the 44/62 protein in formation of the stable holoenzyme DNA replication complex has been further elucidated by specifically examining the role that the 44/62 protein plays in loading the 45 protein onto the DNA substrate. A stable phospho-45 protein or phosphorylated holoenzyme complex was not detected or isolated, suggesting that the 44/62 protein may not act as a protein kinase. Product and dead-end inhibition data are consistent with an ordered kinetic mechanism with respect to product release in which phosphate is released from the 44/62 protein prior to ADP. Positional isotope effect studies support this mechanism and failed to demonstrate that ATP hydrolysis by the 44/62 protein is reversible. Steady-state ATPase assays using aluminum tetrafluoride as an inhibitor are also consistent with release of ADP being partially rate-limiting. Aluminum tetrafluoride acts to trap ADP on the enzyme after turnover, forming a stable transition state analog that dissociates slowly from the enzyme. Processive DNA synthesis does not occur using the accessory proteins in the presence of pre- or post-hydrolysis analogs of ATP nor in the presence of ADP-AlF4, indicating that turnover of the 44/62 protein is absolutely required for formation of the holoenzyme complex. Collectively, data obtained regarding ATP hydrolysis by the 44/62 protein are described in terms of the clamp loading protein functioning as a molecular motor, similar to other systems including myosin and kinesin.