@article {Porcella352567, author = {Sarina Y. Porcella and Natasha C. Koussa and Colin P. Tang and Daphne N. Kramer and Priyanka Srivastava and Duncan J. Smith}, title = {Separable recruitment of DNA Polymerase α for initiation of DNA synthesis at replication origins and lagging-strand priming during replication elongation}, elocation-id = {352567}, year = {2019}, doi = {10.1101/352567}, publisher = {Cold Spring Harbor Laboratory}, abstract = {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.Author summary Half of each eukaryotic genome is replicated continuously as the leading strand, while the other half is synthesized discontinuously as Okazaki fragments on the lagging strand. The bulk of DNA replication is completed by DNA polymerases ε and δ on the leading and lagging strand respectively, while synthesis on each strand is initiated by DNA polymerase α-primase (Pol α). Using the model eukaryote S. cerevisiae, we modulate cellular levels of Pol α and interrogate the impact of this perturbation on both replication initiation on DNA synthesis and cellular viability. We observe that Pol α associates dynamically at the replication fork for initiation on both strands. Although the initiation of both strands is widely thought to be mechanistically similar, we determine that Ctf4, a hub that connects proteins to the replication fork, specifically recruits Pol α to the lagging strand but is not required for leading-strand initiation. We also find that decreased leading-strand initiation results in a checkpoint response that is necessary for viability when Pol α is limiting. Because the DNA replication machinery is highly conserved from budding yeast to humans, this research provides insights into how DNA replication is accomplished throughout eukaryotes.}, URL = {https://www.biorxiv.org/content/early/2019/10/18/352567}, eprint = {https://www.biorxiv.org/content/early/2019/10/18/352567.full.pdf}, journal = {bioRxiv} }