Abstract
The eukaryotic leading strand DNA polymerase (Pol) ε contains 4 subunits, Pol2, Dpb2, Dpb3 and Dpb4. Pol2 is a fusion of two B-family Pols; the N-terminal Pol module is catalytic and the C-terminal Pol module is non-catalytic. Despite extensive efforts, there is no atomic structure for Pol ε holoenzyme, critical to understanding how DNA synthesis is coordinated with unwinding and the DNA path through the CMG helicase-Pol ε-PCNA clamp. We show here a 3.5-Å cryo-EM structure of yeast Pol ε revealing that the Dpb3–Dpb4 subunits bridge the two DNA Pol modules of Pol2, holding them rigid. This information enabled an atomic model of the leading strand replisome. Interestingly, the model suggests that an OB fold in Dbp2 directs leading ssDNA from CMG to the Pol ε active site. These results complete the DNA path from entry of parental DNA into CMG to exit of daughter DNA from PCNA.
Highlights
The eukaryotic leading strand DNA polymerase (Pol) ε contains 4 subunits, Pol[2], Dpb[2], Dpb[3] and Dpb[4]
Pol ε physically associates with the replicative CMG (Cdc[45], Mcm[2,3,4,5,6,7], and GINS) helicase to assemble a molecular machine termed the leading strand replisome that couples continuous DNA unwinding with high fidelity and processive DNA synthesis[8,9,10]
Pol[2] contains two DNA polymerase modules covalently linked in a 2222-residue long polypeptide chain; the catalytic polymerization and proofreading nuclease action are contained in the N-terminal (NTD) module of Pol[2] while the C-terminal (CTD) module of Pol[2] encodes a noncatalytic DNA polymerase that likely serves a structural role[11]
Summary
Dpb[4] subunits each adopt histone folds that form a tight Dpb[3,4] complex[7]. Pol ε physically associates with the replicative CMG (Cdc[45], Mcm[2,3,4,5,6,7], and GINS) helicase to assemble a molecular machine termed the leading strand replisome that couples continuous DNA unwinding with high fidelity and processive DNA synthesis[8,9,10]. The Dpb[2] subunit is essential[14], and studies indicate that it functions with the CTD inactive polymerase module of Pol[2] in assisting initiation factors in the formation of CMG helicase at origins[15,16,17]. Genetic studies reveal that the Dpb[3] and Dpb[4] histone fold subunits are not essential[18,19], but are required for preservation of epigenetic information during replication[20,21]. A most interesting finding is that the active and inactive polymerase modules of Pol[2] are spatially separate and are held together by the Dpb3–Dpb[4] histone fold subunits. The Pol ε structure has enabled us to build a pseudo atomic model of the leading strand replisome, revealing the orthogonal path of the parental DNA entering CMG and the nascent daughter DNA exiting from PCNA, and how the leading single-strand DNA is directed by the Dpb[2] OB domain from the CMG helicase to the Pol ε active site
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
