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Abstract During replication, lagging strand lesions are initially encountered by high-fidelity DNA polymerase (pol) holoenzymes comprised of pol δ and the PCNA sliding clamp. To proceed unhindered, pol δ holoenzymes must bypass lesions without stalling. This entails dNMP incorporation opposite the lesion (insertion) and the 5′ template deoxynucleotide (extension). Historically, it was viewed that high-fidelity pol holoenzymes stall upon encountering lesions, activating DNA damage tolerance pathways that are ultimately responsible for lesion bypass. Our recent study of four prominent lesions revealed that human pol δ holoenzymes support insertion and/or bypass for multiple lesions and the extent of these activities depends on the lesion and pol δ proofreading. In the present study, we expand these analyses to other prominent lesions. Collectively, analyses of 10 lesions from both studies reveal that the insertion and bypass efficiencies of pol δ holoenzymes each span a complete range (0%–100%). Consequently, the fates of pol δ holoenzymes upon encountering lesions are quite diverse. Furthermore, pol δ proofreading promoted holoenzyme progression at 7 of the 10 lesions and did not deter progression at any. Altogether, the results significantly alter our understanding of the replicative capacity of high-fidelity pol holoenzymes and their functional role(s) in lesion bypass.
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Polymerization and editing modes of a high-fidelity DNA polymerase are linked by a well-defined path
Abstract Proofreading by replicative DNA polymerases is a fundamental mechanism ensuring DNA replication fidelity. In proofreading, mis-incorporated nucleotides are excised through the 3′-5′ exonuclease activity of the DNA polymerase holoenzyme. The exonuclease site is distal from the polymerization site, imposing stringent structural and kinetic requirements for efficient primer strand transfer. Yet, the molecular mechanism of this transfer is not known. Here we employ molecular simulations using recent cryo-EM structures and biochemical analyses to delineate an optimal free energy path connecting the polymerization and exonuclease states of E. coli replicative DNA polymerase Pol III. We identify structures for all intermediates, in which the transitioning primer strand is stabilized by conserved Pol III residues along the fingers, thumb and exonuclease domains. We demonstrate switching kinetics on a tens of milliseconds timescale and unveil a complete pol-to-exo switching mechanism, validated by targeted mutational experiments.
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- Award ID(s):
- 2027902
- PAR ID:
- 10226325
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 11
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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null (Ed.)Ribonucleoside monophosphate (rNMP) incorporation in DNA is a natural and prominent phenomenon resulting in DNA structural change and genome instability. While DNA polymerases have different rNMP incorporation rates, little is known whether these enzymes incorporate rNMPs following specific sequence patterns. In this study, we analyzed a series of rNMP incorporation datasets, generated from three rNMP mapping techniques, and obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late firing autonomously replicating sequences (ARS’s) of the yeast genome, from which bidirectional, leading and lagging DNA synthesis starts. We find the preference of rNMP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNMP incorporation changes dramatically within 500 nt from ARS’s, highlighting the Pol δ - Pol ε handoff during early leading-strand synthesis. Furthermore, the pattern of rNMP incorporation is markedly distinct between the leading the lagging strand. Overall, our results show the different counts and patterns of rNMP incorporation during DNA replication from ARS, which reflects the different labor of division and rNMP incorporation pattern of Pol δ and Pol ε.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1038/s41467-020-19165-2
