Tunability of DNA polymerase stability during eukaryotic DNA replication

Abstract

Structural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol α DNA polymerase activity in replication and show that Pol α primase and the lagging-strand Pol δ can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes. Author contributions Conceptualization, J.S.L., L.M.S., G.D.S., M.E.O., A.M.v.O.; Formal analysis, J.S.L., L.M.S., S.H.M.; Funding acquisition, M.E.O., A.M.v.O.; Investigation, J.S.L., L.M.S., G.D.S., S.H.M., V.N., C.M., C.K.; Methodology, J.S.L., L.M.S., A.M.v.O.; Resources, G.D.S., O.Y., G.K.; Software, L.M.S.; Supervision, M.E.O., A.M.v.O.; Validation, J.S.L., L.M.S.; Visualization, J.S.L., L.M.S., A.M.v.O.; Writing -original draft, J.S.L., L.M.S., G.

CUL2LRR1, TRAIP and p97 control CMG helicase disassembly in the mammalian cell cycle

“…The CMG helicase is a metastable complex that is topologically trapped around parental ssDNA throughout the elongation phase of DNA replication (Lewis et al , 2020). Chromatin is disrupted ahead of the CMG helicase and non‐nucleosomal proteins are displaced, without breaking the association of CMG with replication fork DNA (Kurat et al , 2017; Sparks et al , 2019).…”

A hand-off of DNA between archaeal polymerases allows high-fidelity replication to resume at a discrete intermediate three bases past 8-oxoguanine

“…This configuration allows TLS to be transiently available for subsequent encounters with DNA lesions. Yet, in the context of a more complex multi-equilibria system, dynamic exchange of HiFi Pols and other DNA processing enzymes has been observed (53)(54)(55). PCNA123 has also been shown to coordinate an alternate arrangement of enzymes, optimizing their combined activities for Okazaki fragment maturation (56).…”

DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

“…10 7 cells were suspended in buffer A (10 mM HEPES at pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 1 mM DTT, 10 mM NaF, 1 mM sodium orthovanadate, 0.1% Triton X-100, with protease and phosphatase inhibitors) and incubated for 5 mins on ice. Nuclei were recovered by centrifugation at 1300 g for 4 min.…”

“…Eukaryotic replisomes are multiprotein complexes consisting, minimally, of the CMG helicase [MCM2-7 (M), CDC45 (C), and GINS (go, ichi, ni, san) proteins (G)] which forms a ring around the leading strand template. Other components include the pol α, ε, and δ polymerases, MCM10, and a few accessory factors 1–7 . The identification and characterization of the minimal components of biochemically active replisomes, the result of decades of extraordinary work from multiple laboratories, necessarily reflects studies with deproteinized model DNA substrates under carefully controlled conditions.…”