Replication slippage of the thermophilic DNA polymerases B and D from the Euryarchaeota Pyrococcus abyssi.

Melissa Castillo-Lizardo,Ghislaine Henneke,Enrique Viguera

doi:10.3389/fmicb.2014.00403

Abstract

Replication slippage or slipped-strand mispairing involves the misalignment of DNA strands during the replication of repeated DNA sequences, and can lead to genetic rearrangements such as microsatellite instability. Here, we show that PolB and PolD replicative DNA polymerases from the archaeal model Pyrococcus abyssi (Pab) slip in vitro during replication of a single-stranded DNA template carrying a hairpin structure and short direct repeats. We find that this occurs in both their wild-type (exo+) and exonuclease deficient (exo-) forms. The slippage behavior of PabPolB and PabPolD, probably due to limited strand displacement activity, resembles that observed for the high fidelity P. furiosus (Pfu) DNA polymerase. The presence of PabPCNA inhibited PabPolB and PabPolD slippage. We propose a model whereby PabPCNA stimulates strand displacement activity and polymerase progression through the hairpin, thus permitting the error-free replication of repetitive sequences.

Highlights

Low complexity DNA sequences such as microsatellites (1–9 nt repeat length), including mono, di, and trinucleotide repeats, and minisatellites are frequently associated with mutagenesis “hot-spots” in both eukaryotic and prokaryotic genomes (Bierne et al, 1991; Michel, 2000; Aguilera and Gomez-Gonzalez, 2008)
PabPCNA can be loaded onto DNA in the absence of the clamp-loader replication factor C (RF-C), the presence of this factor does enhance its loading (Rouillon et al, 2007). We report that both P. abyssi DNA polymerases slip in vitro on a template that consists of single-stranded DNA with a hairpin structure flanked by short direct repeats
EXPERIMENTAL SYSTEM To study whether P. abyssi thermostable DNA polymerases (Pab pols) promote replication slippage, we performed primerextension assays using the circular single-stranded DNA (ssDNA) template, FXc (Canceill and Ehrlich, 1996; Canceill et al, 1999; Viguera et al, 2001a,b)

Summary

INTRODUCTION

Low complexity DNA sequences such as microsatellites (1–9 nt repeat length), including mono, di, and trinucleotide repeats, and minisatellites (unit ≥10 nt) are frequently associated with mutagenesis “hot-spots” in both eukaryotic and prokaryotic genomes (Bierne et al, 1991; Michel, 2000; Aguilera and Gomez-Gonzalez, 2008) These types of sequences are characterized by high instability, consisting of the addition or deletion of repeated units, leading to variations in repeat copy number. In the context of the model proposed for slippagemediated deletions (Figure 1), a polymerase with high strand displacement activity would be able to open the hairpin duplex, avoiding the polymerase dissociation and nascent strand reannealing steps, and replicate the repeat-containing template faithfully. These results help toward understanding the dynamics of replication through common non-B DNA structures and identifying the key DNA polymerases involved in replication slippage; a crucial step for understanding genome stability in these organisms

MATERIALS AND METHODS

RESULTS

DISCUSSION