Structural basis for processive DNA synthesis by yeast DNA polymerase ɛ

Abstract

DNA polymerase ɛ (Pol ɛ) is a high-fidelity polymerase that has been shown to participate in leading-strand synthesis during DNA replication in eukaryotic cells. We present here a ternary structure of the catalytic core of Pol ɛ (142 kDa) from Saccharomyces cerevisiae in complex with DNA and an incoming nucleotide. This structure provides information about the selection of the correct nucleotide and the positions of amino acids that might be critical for proofreading activity. Pol ɛ has the highest fidelity among B-family polymerases despite the absence of an extended β-hairpin loop that is required for high-fidelity replication by other B-family polymerases. Moreover, the catalytic core has a new domain that allows Pol ɛ to encircle the nascent double-stranded DNA. Altogether, the structure provides an explanation for the high processivity and high fidelity of leading-strand DNA synthesis in eukaryotes.

Modulation of DNA Polymerase Noncovalent Kinetic Transitions by Divalent Cations

“…These transitions include (i) the primer strand transfer between the polymerase and exonuclease sites, (ii) the translocation fluctuations, in which the DNA substrate moves between the pretranslocation and post-translocation states in the DNAP polymerase site, a spatial displacement of the distance of a single nucleotide, and (iii) dNTP binding in the polymerase site. In contrast to the roles of the Me 2ϩ ions in the chemical steps of dNTP polymerization and exonucleolysis, much less is known about the effects of Me 2ϩ ions on these noncovalent transitions in DNAP complexes.The conserved architecture of the DNAP domain that contains the polymerase active site resembles a partially closed hand, comprising palm, thumb, and fingers subdomains (3,4,9,14,15). The palm subdomain contains residues that participate in catalysis of phosphodiester bond formation, including the acidic residues involved in Me 2ϩ coordination.…”

“…The conserved architecture of the DNAP domain that contains the polymerase active site resembles a partially closed hand, comprising palm, thumb, and fingers subdomains (3,4,9,14,15). The palm subdomain contains residues that participate in catalysis of phosphodiester bond formation, including the acidic residues involved in Me 2ϩ coordination.…”

“…This editing reaction allows for removal of incorrect nucleotides inserted during polymerization, contributing ϳ1-2 orders of magnitude to replication fidelity (1). Exonucleolytic editing occurs in a separate active site that is typically ϳ30 -40 Å from the polymerase site (3)(4)(5)(6)(7)(8)(9), and it requires that ϳ3 base pairs of the nascent primertemplate duplex be melted to allow the primer strand to be transferred from the polymerase site to the exonuclease site. An essential role for two divalent metal cations (Me 2ϩ ions) in the mechanism of numerous enzyme-catalyzed nucleotidyl transfer reactions has been characterized (10), in which one Me 2ϩ ion (termed metal A) serves primarily to activate the nucleophile, whereas the other (termed metal B) mitigates the negative charge that builds in the transition state (10,11).…”

“…The B family includes pol ⑀ and pol ␦, the enzymes that catalyze leading and lagging strand genomic replication, respectively, in eukaryotes (18,19). Core structure, catalytic mechanisms, and functional properties are highly conserved in this DNAP family (2,3,8,9,20,21). Based upon the comparison of crystal structures of the DNAP from bacteriophage ⌽29 in the fingers-open, post-translocation state DNAP-DNA binary complex or in the fingers-closed, post-translocation state DNAP-DNA-dNTP ternary complex (3), it was proposed that the post-translocation state ternary complex can serve as a model for the structure of a fingers-closed, pre-translocation state complex.…”

GREM1 and POLE variants in hereditary colorectal cancer syndromes

“…The variant is present in the ExAC database at a frequency of 0.003%. In an attempt to try to understand if the amino‐acid substitution could confer a functional effect on the protein, in silico prediction based on the yeast DNA polymerase, polE, structure (4M8O.pdb), was performed (Hogg et al, 2014). The region of interest exhibits high amino‐acid identity.…”