Deeper understanding of coordinated DNA polymerase and helicase activities during DNA replication achieved with help from Sapphire

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In recent work published in Nature Communications, Xu et al outlined a mechanism for polymerase-helicase coupling based on structural and biochemical studies. Their work examined a yeast replisome using cryo-electron microscopy and found a dynamic mechanism in which the polymerase engages and disengages from the helicase. The authors examined the effect of helicase-polymerase coupling on the enzymatic activities of each, using assays whose output was measured by phosphor imaging on an Azure Sapphire Biomolecular Imager.

Since the release of this publication, the first generation Sapphire used has been succeeded by the new Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more about this new imager.

Phosphor imaging is a method to detect radioactive material in applications such as NorthernSouthern, or Western blotting, or in radiolabeled tissue sections.

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During cell division, DNA must be replicated accurately while maintaining any epigenetic information marked on the parent DNA. DNA replication is carried out by the replisome, a multi-protein complex comprised of a DNA helicase that unwinds the DNA, at least two DNA polymerases that synthesize the new DNA strands, and numerous additional protein factors. For accurate and efficient DNA replication, the helicase and DNA polymerase activities must be coordinated, but how this coordination occurs is not well understood.

How cryo-electron microscopy structures led to models for DNA translocation

In eukaryotes, the DNA helicase is a multi-subunit complex made up of a hexameric helicase shaped like a ring (referred to as MCM) combined with Cdc45 and the GINS complex, together called the CMG complex. Xu et al studied a yeast replisome consisting of the CMG complex, the leading strand polymerase Polε, and accessory factors bound to a DNA replication fork. Cryo-electron microscopy structures revealed Pole cycling on and off the MCM ring as DNA translocated through the helicase. Based on these results, the authors propose a model that may synthesize two opposing models for DNA translocation through the helicase that have been put forward based on prior sets of data. These earlier models state that either

  1. ssDNA threads around the MCM pore in a symmetrical, rotary mechanism, or
  2. that the ssDNA moves through the pore in a nonsymmetric, “inch worm” fashion.

The new experiments suggest that the MCM subunits remain in a planar ring, not an open spiral, and that DNA threads through in a rotary fashion, involving only some ATPase domains per cycle.

What affects the activity of either enzyme?

The authors also examined whether the coupling of helicase and Polε affected the activity of either enzyme (Figure 6). Previous studies have shown that Polε coupling with CMG can cause the helicase to stall at barriers on the DNA fork. Xu et al assayed the helicase activity of the reconstituted yeast replicase in vitro. Components of the assay were separated by native page and 32P-labeled products detected by phosphor imaging on the Sapphire. The results showed that, in this system, the helicase could unwind DNA in the absence of Polε. Adding Polε had little effect on helicase activity, causing only slight suppression. The authors state their structure suggests Polε stabilizes the replisome in a state such that it can stall while fork barriers are removed or repaired.

Phosphor imaging gels using Sapphire Imager from Azure Biosystems
Figure 6 from Xu et al, Synergism between CMG helicase and leading strand DNA polymerase at replication fork, showing helicase (b,c) and replication (g) assays, analyzed by imaging of gels by phosphor imaging on the Sapphire Biomolecular Imager. Licensed under CC BY 4.0.

DNA polymerase activity was also assessed in an in vitro replication assay: 32P-labeled products were separated on a 1% alkaline agarose gel and detected by phosphor imaging on the Sapphire. A mutation in Polε that disrupts coupling with MCM prevented the synthesis of the expected DNA product, suggesting that coupling is required for Polε to carry out leading strand DNA replication.

Since this paper was published, the Sapphire has been succeed by the Sapphire FL

Designed for flexible choice in detection chemistry and samples, the Sapphire FL brings precise quantitation of nucleic acids and proteins
Scientist changing optical modules on the new Azure Sapphire FL
The Azure Sapphire FL Biomolecular Imager is capable of high-resolution imaging and wide depth of field enable many sample types, including arrays, microarrays, Western blots, tissue slides, and small animals.

What a flexible pole hinge region may allow Polε to do

In conclusion, this recent work illuminates how Polε activity is synchronized with DNA translocation by CMG. The authors find that the interaction is dynamic, with Polε cycling on and off of the MCM ring. In their proposed model, a flexible Polε hinge region may allow Pole to flip out of the way to allow for repair of DNA damage or for histone transfer to maintain epigenetic information. Future experiments can investigate the potential role of Polε-MCM coupling in epigenetic inheritance and in replication fork stalling to allow DNA repair.

More about system used in this study

The Sapphire FL is a fluorescent imaging system that includes phosphor imaging among its multiple available imaging applications. Its ability to phosphor image comes in handy especially for applications such as electrophoretic mobility shift assays (EMSA), enzyme assays, and in-vivo imaging. In addition to phosphor imaging, the Sapphire FL can carry out white light, multiplex fluorescence, bioluminescence, and chemiluminescence imaging of a wide variety of sample types, from membranes to slides to model organisms. Learn more about the Sapphire FL here.

SOURCE
  1. Xu Z, Feng J, Yu D, et al. Synergism between CMG helicase and leading strand DNA polymerase at replication fork. Nat Commun. 2023;14:5849.

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