Determining the effects of DNA sequence on Hel308 helicase translocation along single-stranded DNA using nanopore tweezers

Abstract

Motor enzymes that process nucleic-acid substrates play vital roles in all aspects of genome replication, expression, and repair. The DNA and RNA nucleobases are known to affect the kinetics of these systems in biologically meaningful ways. Recently, it was shown that DNA bases control the translocation speed of helicases on single-stranded DNA, however the cause of these effects remains unclear. We use single-molecule picometer-resolution nanopore tweezers (SPRNT) to measure the kinetics of translocation along single-stranded DNA by the helicase Hel308 from Thermococcus gammatolerans. SPRNT can measure enzyme steps with subangstrom resolution on millisecond timescales while simultaneously measuring the absolute position of the enzyme along the DNA substrate. Previous experiments with SPRNT revealed the presence of two distinct substates within the Hel308 ATP hydrolysis cycle, one [ATP]-dependent and the other [ATP]-independent. Here, we analyze in-depth the apparent sequence dependent behavior of the [ATP]-independent step. We find that DNA bases at two sites within Hel308 control sequence-specific kinetics of the [ATP]-independent step. We suggest mechanisms for the observed sequence-specific translocation kinetics. Similar SPRNT measurements and methods can be applied to other nucleic-acid-processing motor enzymes.

Infinite re-reading of single proteins at single-amino-acid resolution using nanopore sequencing

“…The complementary strand has a poly-T 20 tail with a 3’ cholesterol, which associates with the bilayer and increases the concentration of analyte near the pore, improving the event rate( 7, 24, 25 ). For sequencing, we used the mutant nanopore M2 MspA( 26 ) with a cup-like shape( 27 ) that separates the anchoring enzyme by ~10 nm from the constriction of the pore where the blockage of ion current occurs ( 28 ). For the DNA-translocating motor enzyme, we used Hel308 DNA helicase because its observable half-nucleotide ~0.33 nm steps, when pulling ssDNA through MspA( 23 ), are approximately the same as single-amino acid steps.…”

Archaeal DNA Repair Mechanisms

“…The induced and natural competency of many archaeal species permit genetic manipulations, most dependent on HR-directed gene conversion and integrations of new DNA. The archaeal Hel308 enzyme, believed to be responsible for strand displacement during SDSA, has been extensively studied for use in nanopore sequencing [67]. DSB repair is also an essential process for CRISPR viral defense systems found in~85% of Archaea, in which Cas enzymes generate guided double-strand breaks which are subsequently repaired by non-HR DSB repair pathways, NHEJ, and MMEJ [26].…”

Playing a long game

“…For example, Roche has been quietly developing a protein nanopore-based technology acquired from startup Genia as a potential tool for clinical diagnostics, and Ontera is working on a handheld device that uses solid-state nanopores that can potentially identify nucleic acids, proteins, and even pathogens present in a given sample. And at the University of Washington, Jens Gundlach's team has been using nanopore proteins derived from the microbe Mycobacterium smegmatis to study the dynamic interplay between nucleic acids and various 'motor proteins' , such as the helicase enzymes that unwind DNA 15 .…”