Extracts for Analysis of DNA Replication in a Nucleus-Free System

Cold Spring Harbor Protocols
Justin Sparks, Johannes C Walter

Abstract

Frog egg extracts represent a powerful approach with which to dissect molecular mechanisms of vertebrate DNA replication and repair. In the classical approach, sperm chromatin is added to a crude egg lysate to form replication-competent nuclei. We subsequently described a procedure that bypasses the requirement for nuclear assembly in DNA replication. In this method, DNA is first added to a high-speed supernatant (HSS) of egg lysate, which mimics the G1 phase of the cell cycle in that it supports replication licensing. Subsequent addition of a concentrated nucleoplasmic extract (NPE) leads to replication initiation followed by a single complete round of DNA replication. The advantage of the nucleus-free system is that it supports efficient replication of model DNA templates such as plasmids and lambda DNA that can be modified with specific features such as LacI arrays, DNA protein cross-links, or DNA interstrand cross-links. Here, we describe our current protocol for preparation of HSS and NPE. Methods for their use in DNA replication and repair are described elsewhere.

References

May 1, 1988·Proceedings of the National Academy of Sciences of the United States of America·M J LohkaJ L Maller
Jul 14, 1998·Molecular Cell·J WalterJ Newport
Nov 5, 2003·Proceedings of the National Academy of Sciences of the United States of America·Tatyana A ProkhorovaJohannes C Walter
Nov 10, 2004·Gene Expression Patterns : GEP·Brian E Walter, Jonathan J Henry
Sep 23, 2008·Cell·Markus RäschleJohannes C Walter
Jul 1, 2009·Methods in Molecular Biology·Ronald LebofskyJohannes C Walter
Sep 4, 2012·Methods in Molecular Biology·Puck KnipscheerJohannes C Walter
Oct 11, 2014·Cell·Julien P DuxinJohannes C Walter
Sep 1, 2015·Nature·James M DewarJohannes C Walter
Jul 13, 2016·ELife·Yoshitaka KawasoeTatsuro S Takahashi
Apr 22, 2017·Developmental Biology·Wouter S HoogenboomPuck Knipscheer

❮ Previous
Next ❯

Citations

Aug 29, 2018·Cold Spring Harbor Protocols·Susannah Rankin
Sep 19, 2020·Genes & Development·Emily LowJohannes C Walter
Jan 24, 2021·Molecular Cell·Kyle B VrtisJohannes C Walter
Dec 16, 2020·Molecular Cell·Irene GallinaJulien P Duxin
Apr 28, 2021·Nucleic Acids Research·Yu TaoJean Gautier
Sep 25, 2021·Science Advances·Koichi SatoPuck Knipscheer

❮ Previous
Next ❯

Related Concepts

Related Feeds

Related Papers

Methods in Molecular Biology
Ronald LebofskyJohannes C Walter
Methods : a Companion to Methods in Enzymology
Patrick J LupardusKarlene A Cimprich
Cancer Treatment and Research
Randy J Legerski, Christopher Richie
© 2022 Meta ULC. All rights reserved