Effect of SSB binding modes on the slippage error produced by T7 DNA polymerase during replication of DNA repeated sequences

Abstract

Arrest of the replication machinery within a repeated region is associated with the instability of DNA repeat regions. Primer-template misalignment, also known as replication slippage or copy-choice recombination, is one of the mechanisms involved in the generation of deletions or expansions within repeat regions1,2. A direct role for replication slippage in the deletion of repeated sequences at a hairpin structure has been demonstrated both in vivo and in vitro. Blockage of the DNA polymerase at a hairpin structure followed by polymerase dissociation and transient out-of-frame re-annealing of nascent and template strands cause slipped strand mispairing, generating a deletion on the nascent strand1.

Several DNA polymerases have been tested for their propensity to slip in vitro while replicating hairpin-containing templates3. Studies on DNA polymerases involved in DNA repair such as E. coli Pol I, E. coli Pol II and replicative DNA polymerases such as E. coli Pol III HE or the T4, T7 and F29 phage DNA polymerases revealed that the strand displacement activity (sda) of a DNA polymerase is inversely related to their propensity to slip3.

We have analyzed the role of the SSB protein from E. coli on the slippage performed by T7 DNA polymerase. Slippage is inhibited at increasing SSB concentrations as SSB stimulates specifically the sda of T7 DNA polymerase. Reactions conditions that promote the transition between the (SSB)56 to (SSB)65 binding modes correlate with an specific decrease of the sda of T7 DNA polymerase, thus generating slippage errors. No effect of the SSB binding modes was found on the slippage errors performed by T4 DNA pol on the same DNA template, suggesting that this stimulation seems to be specific for T7 DNA polymerase.

References 1. E. Viguera, D. Canceill, and S.D. Ehrlich, Embo J. Replication slippage involves DNA polymerase pausing and dissociation, 2001, 20, 2587-2595. 2. S.T. Lovett, Mol. Microbiol. Encoded errors: mutations and rearrangements mediated by misalignment at repetitive DNA sequences, 2004, 52: 1243-53. 3. D. Canceill, E. Viguera and S.D. Ehrlich, J. Biol. Chem. Replication slippage of different DNA polymerases is inversely related to their strand displacement efficiency, 1999, 274, 27481-27490.