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| Title | CryoEM insights into RNA primer synthesis by the human primosome. |
|---|---|
| Journal, issue, pages | FEBS J, Vol. 291, Issue 8, Page 1813-1829, Year 2024 |
| Publish date | Feb 9, 2024 |
 Authors | Zhan Yin / Mairi L Kilkenny / De-Sheng Ker / Luca Pellegrini /  |
| PubMed Abstract | Eukaryotic DNA replication depends on the primosome - a complex of DNA polymerase alpha (Pol α) and primase - to initiate DNA synthesis by polymerisation of an RNA-DNA primer. Primer synthesis ...Eukaryotic DNA replication depends on the primosome - a complex of DNA polymerase alpha (Pol α) and primase - to initiate DNA synthesis by polymerisation of an RNA-DNA primer. Primer synthesis requires the tight coordination of primase and polymerase activities. Recent cryo-electron microscopy (cryoEM) analyses have elucidated the extensive conformational transitions required for RNA primer handover between primase and Pol α and primer elongation by Pol α. Because of the intrinsic flexibility of the primosome, however, structural information about the initiation of RNA primer synthesis is still lacking. Here, we capture cryoEM snapshots of the priming reaction to reveal the conformational trajectory of the human primosome that brings DNA primase subunits 1 and 2 (PRIM1 and PRIM2, respectively) together, poised for RNA synthesis. Furthermore, we provide experimental evidence for the continuous association of primase subunit PRIM2 with the RNA primer during primer synthesis, and for how both initiation and termination of RNA primer polymerisation are licenced by specific rearrangements of DNA polymerase alpha catalytic subunit (POLA1), the polymerase subunit of Pol α. Our findings fill a critical gap in our understanding of the conformational changes that underpin the synthesis of the RNA primer by the primosome. Together with existing evidence, they provide a complete description of the structural dynamics of the human primosome during DNA replication initiation. |
 External links | FEBS J / PubMed:38335062 |
| Methods | EM (single particle) |
| Resolution | 3.07 - 6.15 Å |
| Structure data | EMDB-17795, PDB-8qj7:  EMDB-17807: Cryo-EM structure of human DNA polymerase alpha-primase in pre-initiation stage 2  EMDB-17810: Cryo-EM structure of human DNA polymerase alpha-primase in pre-initiation stage 3  EMDB-17811: Cryo-EM structure of human DNA polymerase alpha-primase in pre-initiation stage 4  EMDB-17812: Cryo-EM structure of human DNA polymerase alpha-primase at initiation I  EMDB-17813: Cryo-EM structure of human DNA polymerase alpha-primase at initiation II  EMDB-17824: Cryo-EM structure of human DNA polymerase alpha-primase in primer handover |
| Chemicals |  ChemComp-ZN:  ChemComp-MN:  ChemComp-ATP:  ChemComp-SF4: |
| Source |
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 Keywords |  DNA BINDING PROTEIN / DNA polymerase / complex |
