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TitleCompetition for the nascent leading strand shapes the requirements for PCNA loading in the replisome.
Journal, issue, pagesEMBO J, Vol. 44, Issue 8, Page 2298-2322, Year 2025
Publish dateFeb 28, 2025
AuthorsEmma E Fletcher / Morgan L Jones / Joseph T P Yeeles /
PubMed AbstractDuring DNA replication, the DNA polymerases Pol δ and Pol ε utilise the ring-shaped sliding clamp PCNA to enhance their processivity. PCNA loading onto DNA is accomplished by the clamp loaders RFC ...During DNA replication, the DNA polymerases Pol δ and Pol ε utilise the ring-shaped sliding clamp PCNA to enhance their processivity. PCNA loading onto DNA is accomplished by the clamp loaders RFC and Ctf18-RFC, which function primarily on the lagging and the leading strand, respectively. RFC activity is essential for lagging-strand replication by Pol δ, but it is unclear why Ctf18-RFC is required for leading-strand PCNA loading and why RFC cannot fulfil this function. Here, we show that RFC cannot load PCNA once Pol ε has been incorporated into the budding yeast replisome and commenced leading-strand synthesis, and this state is maintained during replisome progression. By contrast, we find that Ctf18-RFC is uniquely equipped to load PCNA onto the leading strand and show that this activity requires a direct interaction between Ctf18 and the CMG (Cdc45-MCM-GINS) helicase. Our work uncovers a mechanistic basis for why replisomes require a dedicated leading-strand clamp loader.
External linksEMBO J / PubMed:40021844 / PubMed Central
MethodsEM (single particle)
Resolution3.34 - 5.98 Å
Structure data

EMDB-52107: S. cerevisiae CMG, Ctf4, T-C, DNA. Local refinement following signal subtraction
Method: EM (single particle) / Resolution: 3.34 Å

EMDB-52116: S. cerevisiae polymerase epsilon and Ctf18-RFC
Method: EM (single particle) / Resolution: 5.98 Å

EMDB-52120: S. cerevisiae polymerase epsilon catalytic domain, Ctf18-RFC complex
Method: EM (single particle) / Resolution: 3.82 Å

EMDB-52459: Budding yeast replisome consensus refinement containing density for the pol-epsilon catalytic module
Method: EM (single particle) / Resolution: 3.82 Å

EMDB-52505: Polymerase epsilon local refinement in the absence of Ctf18-RFC
Method: EM (single particle) / Resolution: 4.25 Å

Source
  • Saccharomyces cerevisiae (brewer's yeast)

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