Structures of phi29 DNA polymerase complexed with substrate: the mechanism of translocation in B-family polymerases

Abstract

Replicative DNA polymerases (DNAPs) move along template DNA in a processive manner. The structural basis of the mechanism of translocation has been better studied in the A-family of polymerases than in the B-family of replicative polymerases. To address this issue, we have determined the X-ray crystal structures of phi29 DNAP, a member of the protein-primed subgroup of the B-family of polymerases, complexed with primer-template DNA in the presence or absence of the incoming nucleoside triphosphate, the pre-and post-translocated states, respectively. Comparison of these structures reveals a mechanism of translocation that appears to be facilitated by the coordinated movement of two conserved tyrosine residues into the insertion site. This differs from the mechanism employed by the A-family polymerases, in which a conserved tyrosine moves into the templating and insertion sites during the translocation step. Polymerases from the two families also interact with downstream single-stranded template DNA in very different ways.

Lesion processing: high-fidelity versus lesion-bypass DNA polymerases

“…Translocation is the processive movement of a polymerase relative to the DNA duplex between two rounds of the phosphoryl-transfer reaction during polynucleotide replication [32]; it is essential for polymerase function and necessarily occurs with every polymerase upon completion of one replication cycle ( Figure 1). Although the actual dynamic mechanism of translocation is not known for any polymerase, static crystal structures can indicate plausible intermediate states in this process.…”

“…By contrast, the BF crystal structures indicate that the entire translocation cycle occurs following dNTP entry into the BF active site and the induced-fit polymerase closing. The driving forces that could power translocation have been discussed based on crystal structures of a B-family DNA polymerase, phi29 from Bacillus subtilis [32]: dissociation of pyrophosphate breaks the electrostatic link between the fingers domain and the catalytic Mg 2+ ions, enabling the fingers to pivot to the open position. Then, two conserved tyrosine residues enter the insertion site and sterically exclude the nascent base pair.…”

DNA polymerase from temperate phage Bam35 is endowed with processive polymerization and abasic sites translesion synthesis capacity

“…This mismatch-induced stalling, often referred to as "short-term memory," is a consequence of long-range distortions in the DNA that affect the conformation of the protein active site, mainly by template strand-mediated distortions (51). In positions −3 and −4, the template base moves into the minor groove in a conformation that is stabilized by direct contacts between the enzyme and the DNA backbone and solvent-mediated contacts in the minor groove; however, minor groove interactions on Φ29DNAP: DNA complex are stabilized by conserved residues in the thumb subdomain (52,53), thanks to interactions with the primer strand bases or sugar-phosphate backbone up to the −5 position (52). Similarly, the thumb subdomain of B35DNAP would provide interactions with the mismatched wobble up to one helix turn, providing a "memory" during stepwise polymerization.…”

A DNA Nanotransport Device Powered by Polymerase ϕ29

“…We use φ29, a polymerase known for its exceptional strand displacement activity, to push a DNA cargo. Researchers have studied the structure of φ29 polymerase and have provided useful insights into its exceptional strand displacement and processivity, and have deduced its translocation mechanism [9,16,17,32].…”