Mapping and mutation of the conserved DNA polymerase interaction motif (DPIM) located in the C-terminal domain of fission yeast DNA polymerase δ subunit Cdc27

Here it is shown that the catalytic subunit of Pol α, Pol1, interacts with Cdc27, one of three non-catalytic subunits of fission yeast Pol δ, both in vivo and in vitro. Pol1 interacts with the C-terminal domain of Cdc27, at a site distinct from the previously identified binding sites for Cdc1 and PCNA. Comparative protein sequence analysis identifies a protein sequence motif, called the DNA polymerase interaction motif (DPIM), in Cdc27 orthologues from a wide variety of eukaryotic species, including mammals. Mutational analysis shows that the DPIM in fission yeast Cdc27 is not required for effective DNA replication, repair or checkpoint function.The absence of any detectable phenotypic consequences arising from mutation of the DPIM suggests that despite its evolutionary conservation, the interaction between the two polymerases mediated by this motif is a non-essential one.Three conserved DNA polymerase enzymes whose activities are essential for complete chromosomal DNA replication have been identified through biochemical studies in mammalian systems [1] and combined genetic and biochemical studies in yeast [2]. During S-phase, the DNA polymerase α-primase complex synthesises the short RNA-DNA segment that is used to prime synthesis of the leading strand at the chromosomal replication origin and synthesis of each Okazaki fragment on the lagging strand. The short RNA segment is synthesised by the primase and then extended by 10–20 nucleotides by Pol α. The 3' end of the RNA-DNA primer is recognised by replication factor C (RFC), which displaces the Pol α-primase complex and catalyses the loading of the sliding clamp PCNA at the primer-template junction. PCNA then acts as a processivity factor for the Pol δ and/or Pol ε enzymes. The exact roles played by Pol δ and Pol ε remain unclear (for a recent perspective, see ref. [3] and references therein) but Pol δ is most likely responsible for lagging strand replication and may also play a role on the leading strand. Yeast l