Single Molecule Studies of the Recognition Sequence Finding Mechanism of Protelomerase Telk

Protelomerase TelK is an enzyme responsible for forming DNA hairpins in linear prokaryotic DNA. The mechanism by which this protein recognizes its target sequence and both quickly and accurately catalyzes DNA hairpin formation is poorly understood. To investigate the target recognition process, we used TIRF microscopy to visualize quantum dot-labeled TelK interacting with both nonspecific DNA and DNA containing the TelK target sequence. While many sequence-specific DNA-binding proteins (SSDBP) have been shown to scan DNA in 1D as their primary method for locating their recognition sequence1, we surprisingly find that TelK does not move laterally on either aforementioned DNA substrate and therefore does not search by 1D scanning. Measurements of a c-terminally truncated TelK mutant reveal the same behavior. Interestingly, this mutant forms DNA hairpins 50 times slower than wild type, and dissociates from nonspecific DNA at a comparably lower rate than full-length TelK. These results suggest that dissociation from nonspecific DNA is an essential step in the recognition sequence search. Complementary studies with high-resolution optical tweezers reveal that TelK binding to DNA is a highly tension dependent process and condenses the molecule by several nanometers, consistent with crystal structures of the protein-DNA complex2. Remarkably, this condensation is observed on nonspecific DNA as well, despite the fact that these DNA distortions are energetically expensive. These findings suggest that the TelK target sequence search may involve 3D hopping and intersegmental transfer in lieu of 1D scanning. This may represent a novel SSDBP recognition sequence search mechanism.1. Halford, S. et al. Nucl. Ac. Res. 32 (2004)2. Aihara, H. et al. Mol. Cell. 27, 901 (2007)