[Pre-Print] Visualizing the Dynamics of DNA Replication and Repair at the Single-Molecule Molecule Level

Scott Berger and Gheorghe Chistol (2022). [PDF]

During cell division, the genome of each eukaryotic cell is copied by thousands of replisomes – large protein complexes consisting of several dozen proteins. Recent studies suggest that the eukaryotic replisome is much more dynamic than previously thought. To directly visualize replisome dynamics in a physiological context, we recently developed a single-molecule approach for imaging replication proteins in Xenopus egg extracts. These extracts contain all the soluble nuclear proteins and faithfully recapitulate DNA replication and repair in vitro, serving as a powerful platform for studying the mechanisms of genome maintenance. Here we present detailed protocols for conducting single-molecule experiments in nuclear egg extracts and preparing key reagents. This workflow can be easily adapted to visualize the dynamics and function of other proteins implicated in DNA replication and repair.

Single-strand DNA breaks cause replisome disassembly

Vrtis K., Dewar J., Chistol G., Wu A.R., Graham T.G.W., Walter J.C. Molecular Cell, 81:1309-18 (2021). [PDF]

The DNA replication fork suppresses CMG unloading from chromatin before termination

Low* E., Chistol* G [@]., Zaher M.S., Kochenova O.V., Walter J.C [@]. Genes & Development, 34:1534-45 (2020). [PDF]

* – these authors contributed equally.
[@] – corresponding authors

TRAIP Is a Master Regulator of DNA Interstrand Crosslink Repair

Wu R.A., Semlow D.R., Kamimae-Lanning A.N., Kochenova O.V., Chistol G. , Hodskinson M.R., Amunugama R., Sparks J.L., Wang M., Deng L., Mimoso C.A., Low E., Patel K.J., Walter J.C. Nature. 567(7747):267-72 (2019).

The CMG Helicase Bypasses DNA-Protein Cross-Links to Facilitate Their Repair

Sparks J.L.*, Chistol G.*, Gao A.O., Räschle M., Larsen N.B., Mann M., Duxin J.P., & Walter J.C. Cell, 176, 167-81.e21 (2019). [PDF]

Mechanochemical Coupling and Bi-Phasic Force-Velocity Dependence in the Ultra-Fast Ring ATPase SpoIIIE

Liu N.*, Chistol G.*, Cui Y. & Bustamante C. eLife, 7 (2018). [PDF]

Two-subunit DNA Escort Mechanism And Inactive Subunit Bypass In An Ultra-Fast Ring ATPase

Liu N.*, Chistol G.* & Bustamante, C. eLife, 4 (2015). [PDF]

[Review] Mechanical Operation and Intersubunit Coordination of Ring-Shaped Molecular Motors: Insights from Single-Molecule Studies

Liu S.*, Chistol G.* & Bustamante C. Biophysical Journal, 106, 1844–58 (2014). [PDF]

[Preview] Molecular Watchdogs on Genome Patrol

Chistol G. & Walter J.C. eLife 3, e02854 (2014). [PDF]

A Viral Packaging Motor Varies Its DNA Rotation and Step Size to Preserve Subunit Coordination as the Capsid Fills

Liu S.*, Chistol G.*, Hetherington C.L.*, Tafoya S., Aathavan K., Schnitzbauer J., Grimes S., Jardine P.J., & Bustamante C. Cell 157, 702–13 (2014). [PDF]

High Degree of Coordination and Division of Labor Among Subunits in a Homomeric Ring ATPase

Chistol G.*, Liu S.*, Hetherington C.L., Moffitt J.R., Grimes S., Jardine P.J., & Bustamante C. Cell 151, 1017–28 (2012). [PDF]

ClpX(P) Generates Mechanical Force to Unfold and Translocate Its Protein Substrates

Maillard R.A., Chistol G., Sen M., Righini M., Tan J., Kaiser C.M., Hodges C., Martin A., & Bustamante C. Cell 145, 459–69 (2011). [PDF]

[Preview] Single-Molecule Visualization of MCM2-7 DNA Loading: Seeing Is Believing

Chistol G. & Walter J.C. Cell 161, 429-30 (2015). [PDF]