Sequence Specific pausing of RecQ Helicase

Abstract

Escherichia coli RecQ helicase is a Super-Family 2 helicase that plays an essential role in the maintenance of genome stability via its participation in the repair and homologous recombination of DNA. The strand separation (unwinding) mechanism of RecQ has not been well characterized to date. Here, we report the study of RecQ unwinding using magnetic tweezers and focus on understanding the sequence specific pausing behavior observed in both dsDNA unwinding and ssDNA translocation. We compare the unwinding and pausing behavior of RecQ on different DNA sequences and in different pulling and unwinding geometries, which allow us to examine how the key unwinding properties (i.e., unwinding rate, processivity, pause location, pause duration, and their distributions) depend on DNA sequence, DNA geometry, and applied tension on the DNA. DNA unwinding by wild-type RecQ helicase is interrupted by strong sequence-dependent pauses. Pausing is significantly reduced for thrombin-cleaved RecQ and truncation constructs lacking the HRDC domain, and for point mutants that disrupt the single-stranded binding affinity of the HRDC domain. We propose a model for the sequence dependent pausing by RecQ and speculate on the in vivo ramifications of this behavior.