Behind the paper: High-temperature single-molecule kinetic analysis of thermophilic archaeal MCM helicases

NEB and NIST scientists devised a single-molecule assay to quantitatively measure long-range DNA unwinding by individual helicase enzymes. Kelly shares details of this recent publication.

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Hi, my name is Kelly Schermerhorn. I'm a research scientist in the DNA Enzyme's Division here at NEB. I'm a member of the Gardner Lab, and we're interested in understanding how organisms of the archaea branch carry out DNA replication.

Our most recent publication was entitled High-Temperature Single-Molecule Kinetic Analysis of Thermophilic MCM helicases, was done in collaboration with Nathan Tanner who's here at NEB, and Zvi Kelman who's at the biomolecular labeling laboratory which is a joint facility owned by the National Institute of Standards and Technology and the University of Maryland.

In this work we set to establish a single molecule technique that would allow us to observe DNA replication enzymes in action in real time. In particular, we're really interested in understanding how the enzymes of the DNA replisome will coordinate with one another to faithfully replicate an organism's genome.

One enzyme involved in that process is a DNA helicase, and this enzyme is responsible for unwinding double stranded DNA, and it does so by using the ATP hydrolysis for energy. Now in the organisms we're interested in, in the Archaeal branch as well as those in the Eukarya, the helicase is known as the Mini Chromosome Maintenance Complex or MCM.

In this study, we looked at MCM from two different species. Now for the MCM helicase single molecule experiments we'll be able to observe a single MCM helicase unwinding a single DNA substrate.  From this, we'll be able to obtain how far the DNA helicase has traveled in base pairs as well as how fast the helicase has traveled in base pairs per second.

First we needed to construct a flow cell, and this was done in-house. Now the flow cell is made up of a biotin functionalized slide and on top of that biotin functionalized slide is a piece of double sided tape that has a double Y shaped channel cut out of it. On top of the double sided tape is a quartz slide that has four holes drilled into it. Now, in these four holes go tubing, and there are two inlet tubes and two outlet tubes. The inlet tubes allow for delivery of the experimental reagents and the outlet tubes allow for removal of the experimental reagents, and on top of the flow cell is an aluminum block and the aluminum block has a hole in the side that houses a cartridge heater.

The cartridge heater is hooked up to a variable autotransformer and this is used to heat the system to 65 degrees so that we can carry out our high temperature experiments. After we've constructed the flow cell we then need to construct a DNA construct which is made from Lambda DNA and four different oligos. Oligo three has a five prime biotin on it, and Oligo four has a reflective magnetic bead. Once the construct is created we deliver it to our flow cell and the five prime biotin will interact with our functionalized slide and tether the DNA construct to the flow cell.

Once the DNA is tethered to the flow cell we're ready to carry out our experiments. DNA is placed under laminar flow under low peak in newton force. In these experiments we're carrying them out at three peak in newtons of force. Now, at this low force, double stranded DNA is stretched out to near its persistence length. While single strand DNA entropically collapses upon itself into a ball. Any enzyme that can convert single stranded DNA to double stranded DNA, or double stranded DNA to single stranded DNA, can be used by this single molecule technique to observe its action.

Interestingly, in five to ten percent of the DNA unwinding events, we observe two successive trajectories; where we think the DNA helicase is unwinding the construct, it's losing it's grip, trans-locating back, regaining its grip and unwinding the construct a second time. Now, this is a feature of DNA helicase's that can only be observed using the single molecule technique. This may provide mechanistic insight to how the DNA helicase coordinates with the replisome in the DNA preliminary string DNA synthesis.

The development and implementation of this high temperature single molecule assay allows us to determine the kinetic parameters for a wide variety of processive DNA enzymes including exonucleases and polymerases. These kinetic parameters really provide insight into how these enzymes carry out their function.

In future work, we hope to determine how the enzymes that are involved in DNA replication in Archaea coordinate with one another to carry out processive DNA synthesis.

For more details, you can visit our open access publication on Nucleic Acids Research.

 

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