Fidelity of DNA replication-a matter of proofreading

Abstract

DNA that is transmitted to daughter cells must be accurately duplicated to maintain genetic integrity and to promote genetic continuity. A major function of replicative DNA polymerases is to replicate DNA with the very high accuracy. The fidelity of DNA replication relies on nucleotide selectivity of replicative DNA polymerase, exonucleolytic proofreading, and postreplicative DNA mismatch repair (MMR). Proofreading activity that assists most of the replicative polymerases is responsible for removal of incorrectly incorporated nucleotides from the primer terminus before further primer extension. It is estimated that proofreading improves the fidelity by a 2-3 orders of magnitude. The primer with the incorrect terminal nucleotide has to be moved to exonuclease active site, and after removal of the wrong nucleotide must be transferred back to polymerase active site. The mechanism that allows the transfer of the primer between pol and exo site is not well understood. While defects in MMR are well known to be linked with increased cancer incidence only recently, the replicative polymerases that have alterations in the exonuclease domain have been associated with some sporadic and hereditary human cancers. In this review, we would like to emphasize the importance of proofreading (3'-5' exonuclease activity) in the fidelity of DNA replication and to highlight what is known about switching from polymerase to exonuclease active site.

Keywords: 3′-5′ proofreading; Fidelity; Polymerase structure; Replicative polymerases.

Fig. 1

Position of the exonuclease and polymerase active sites in A family (Klenow polymerase) and B family (RB69 polymerase). The enzymes are in cartoon representation with the polymerase domain in grey and exonuclease domain in red and DNA in orange. The images were generated using PyMol (DeLano 2002), and are based on the crystal structure of Klenow in the complex with DNA (PDB ID code 1KLN) and the ternary complex structure of RB69 polymerase (PDB ID code 3NCI)

Fig. 2

Movement of fingers domain from the “closed” to “open” conformation in RB69 DNA polymerase. Fingers’ movements brings conserved residues from motif B closer to palm catalytic residues D411 and D623 creating polymerase active site. The image was created using PyMol (DeLano 2002) and the ternary complex structure of RB69 polymerase (PDB ID code 3NCI)

Fig. 3

Position of the β-hairpin loop in editing (a) and replicating (b) modes. Superposition of the two structures showing the movement of the β-hairpin loop (c). The images were generated using PyMol (DeLano 2002) based on the ternary crystal structure of RB69 DNA polymerase (PDB ID code 3NCI) and editing structure (PDB ID 1CLQ)