Separable recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation

During eukaryotic DNA replication, DNA polymerase alpha/primase (Pol α) initiates synthesis on both the leading and lagging strands. It is unknown whether leading- and lagging-strand priming are mechanistically identical, and whether Pol α associates processively or distributively with the replisome. Here, we titrate cellular levels of Pol α in S. cerevisiae and analyze Okazaki fragments to study both replication initiation and ongoing lagging-strand synthesis in vivo. We observe that both Okazaki fragment initiation and the productive firing of replication origins are sensitive to Pol α abundance, but find that the absence of the replisome adaptor protein Ctf4 only impairs lagging-strand initiation. Our results suggest that distinct modes of Pol α recruitment exist for replication initiation and elongation, and are consistent with distributive association of Pol α with the replisome. Additionally, we observe that activation of the checkpoint becomes essential for viability upon severe depletion of Pol α, and demonstrate that this checkpoint requirement is due to impaired origin firing as opposed to reduced lagging-strand priming.