Abstract
DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Here we report the ternary structure of the Polε catalytic subunit (Pol2) bound to a nascent G:C base pair (Pol2G:C). Pol2G:C has a typical B-family polymerase fold and embraces the template-primer duplex with the palm, fingers, thumb and exonuclease domains. The overall arrangement of domains is similar to the structure of Pol2T:A reported recently, but there are notable differences in their polymerase and exonuclease active sites. In particular, we observe Ca2+ ions at both positions A and B in the polymerase active site and also observe a Ca2+ at position B of the exonuclease site. We find that the contacts to the nascent G:C base pair in the Pol2G:C structure are maintained in the Pol2T:A structure and reflect the comparable fidelity of Pol2 for nascent purine-pyrimidine and pyrimidine-purine base pairs. We note that unlike that of Pol3, the shape of the nascent base pair binding pocket in Pol2 is modulated from the major grove side by the presence of Tyr431. Together with Pol2T:A, our results provide a framework for understanding the structural basis of high fidelity DNA synthesis by Pol2.
Highlights
The bulk of DNA synthesis in eukaryotes is carried out by three polymerases: Pols a, d, and e [1,2]
We present here a crystal structure of yeast Pol2 catalytic domain that differs from the Pol2T:A structure in containing a slightly different protein construct, a different template-primer, a different incoming nucleotide, a different nascent base pair (G:C versus T:A), and different metals (Ca2+ versus Mg2+)
Our attempts to determine the structure by molecular replacement (MR) methods using the Pol3 structure as a search model resulted in a satisfactory MR solution
Summary
The bulk of DNA synthesis in eukaryotes is carried out by three polymerases: Pols a, d, and e [1,2]. Pole is believed to be the leading strand polymerase and, like Pold, achieves fidelity via both accurate DNA polymerization and 39R59 proofreading exonuclease (Exo) activities. The DNA polymerization (Pol) activities of Pols d and e achieve an error rate of ,1025, which is further lowered to ,1027 by their proofreading functions. DNA mismatch repair achieves another ,100-fold increase in fidelity, for an error rate of ,1029 following DNA synthesis. Accurate DNA replication by Pols d and e is crucial in maintaining genome integrity and mutations that lower the fidelity of these polymerases lead to tumor development. Germline and somatic mutations in the exonuclease domains of Pols d and e are frequently associated with endometrial and colon cancers [3,4,5,6]
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 call
