Abstract
The B-subunit (p70/Pol12p) of the DNA polymerase alpha-primase (Polalpha-primase) complex is thought to have a regulatory role in an early stage of S phase. We generated a panel of fission yeast thermosensitive mutants of the B-subunit (termed Spb70) to investigate its role in initiation of DNA replication by genetic and biochemical approaches. Here, we show that the fission yeast Spb70 genetically interacts and coprecipitates with origin recognition complex proteins Orp1/Orc1 and Orp2/Orc2 and primase coupling subunit Spp2/p58. A fraction of Spb70 associates with Orp2 on chromatin throughout the cell cycle independent of the other subunits of Polalpha-primase. Furthermore, primase Spp2/p58 subunit preferentially associates with the unphosphorylated Orp2, and the association requires Spb70. Mutations in orp2+ that abolish or mimic the Cdc2 phosphorylation of Orp2 suppress or exacerbate the thermosensitivity of the spb70 mutants, respectively, indicating that an unphosphorylated Orp2 promotes an Spb70-dependent replication event. Together, these results indicate that the chromatin-bound B-subunit in association with origin recognition complex mediates recruiting Polalpha-primase complex onto replication origins in G1 pre-Start through an interaction with primase Spp2/p58 subunit. Our results thus suggest a role for the recruited Polalpha-primase in the initiation of both leading and lagging strands at the replication origins.