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

biorxiv. 2019; 
Sarina Y. Porcella,  Natasha C. Koussa,  Colin P. Tang,  Daphne N. Kramer,  Priyanka Srivastava, Duncan J. Smith
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Recombinant Proteins Samples were transferred to PVDF, blocked with 5% milk and probed with C-Myc antibody (Genscript A00173-100), Rad53 antibody (Abcam ab104232), histone H4 antibody (Abcam ab10158), or actin antibody (Thermofisher Scientific MA1-744) Get A Quote

摘要

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... More

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