Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Methods
2007 Feb 01;412:222-31. doi: 10.1016/j.ymeth.2006.07.024.
Show Gene links
Show Anatomy links
Analyzing the ATR-mediated checkpoint using Xenopus egg extracts.
Lupardus PJ
,
Van C
,
Cimprich KA
.
???displayArticle.abstract???
Our knowledge of cell cycle events such as DNA replication and mitosis has been advanced significantly through the use of Xenopus egg extracts as a model system. More recently, Xenopus extracts have been used to investigate the cellular mechanisms that ensure accurate and complete duplication of the genome, processes otherwise known as the DNA damage and replication checkpoints. Here we describe several Xenopus extract methods that have advanced the study of the ATR-mediated checkpoints. These include a protocol for the preparation of nucleoplasmic extract (NPE), which is a soluble extract system useful for studying nuclear events such as DNA replication and checkpoints. In addition, we describe several key assays for studying checkpoint activation as well as methods for using small DNA structures to activate ATR.
Abraham,
Cell cycle checkpoint signaling through the ATM and ATR kinases.
2001, Pubmed
Abraham,
Cell cycle checkpoint signaling through the ATM and ATR kinases.
2001,
Pubmed
Arias,
Initiation of DNA replication in Xenopus egg extracts.
2004,
Pubmed
,
Xenbase
Blow,
Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs.
1986,
Pubmed
,
Xenbase
Byun,
Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.
2005,
Pubmed
,
Xenbase
Chen,
Chk1 in the DNA damage response: conserved roles from yeasts to mammals.
2004,
Pubmed
Cimprich,
Fragile sites: breaking up over a slowdown.
2003,
Pubmed
Costanzo,
An ATR- and Cdc7-dependent DNA damage checkpoint that inhibits initiation of DNA replication.
2003,
Pubmed
,
Xenbase
Felix,
A post-ribosomal supernatant from activated Xenopus eggs that displays post-translationally regulated oscillation of its cdc2+ mitotic kinase activity.
1989,
Pubmed
,
Xenbase
Guo,
Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts.
2000,
Pubmed
,
Xenbase
Hekmat-Nejad,
Xenopus ATR is a replication-dependent chromatin-binding protein required for the DNA replication checkpoint.
,
Pubmed
,
Xenbase
Hutchison,
Periodic DNA synthesis in cell-free extracts of Xenopus eggs.
1987,
Pubmed
,
Xenbase
Jones,
XRad17 is required for the activation of XChk1 but not XCds1 during checkpoint signaling in Xenopus.
2003,
Pubmed
,
Xenbase
Kumagai,
The Xenopus Chk1 protein kinase mediates a caffeine-sensitive pathway of checkpoint control in cell-free extracts.
1998,
Pubmed
,
Xenbase
Lee,
Claspin, a Chk1-regulatory protein, monitors DNA replication on chromatin independently of RPA, ATR, and Rad17.
2003,
Pubmed
,
Xenbase
Liu,
Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint.
2000,
Pubmed
Lohka,
Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components.
1983,
Pubmed
,
Xenbase
Lohka,
Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts.
1985,
Pubmed
,
Xenbase
Luciani,
Characterization of a novel ATR-dependent, Chk1-independent, intra-S-phase checkpoint that suppresses initiation of replication in Xenopus.
2004,
Pubmed
,
Xenbase
Lupardus,
A requirement for replication in activation of the ATR-dependent DNA damage checkpoint.
2002,
Pubmed
,
Xenbase
Lupardus,
Phosphorylation of Xenopus Rad1 and Hus1 defines a readout for ATR activation that is independent of Claspin and the Rad9 carboxy terminus.
2006,
Pubmed
,
Xenbase
McGarry,
Geminin, an inhibitor of DNA replication, is degraded during mitosis.
1998,
Pubmed
,
Xenbase
Melo,
A unified view of the DNA-damage checkpoint.
2002,
Pubmed
,
Xenbase
Michael,
Activation of the DNA replication checkpoint through RNA synthesis by primase.
2000,
Pubmed
,
Xenbase
Murray,
Cyclin synthesis drives the early embryonic cell cycle.
1989,
Pubmed
,
Xenbase
Murray,
The role of cyclin synthesis and degradation in the control of maturation promoting factor activity.
1989,
Pubmed
,
Xenbase
Murray,
Cell cycle extracts.
1991,
Pubmed
Méchali,
DNA synthesis in a cell-free system from Xenopus eggs: priming and elongation on single-stranded DNA in vitro.
1982,
Pubmed
,
Xenbase
Newport,
Nuclear reconstitution in vitro: stages of assembly around protein-free DNA.
1987,
Pubmed
,
Xenbase
Pacek,
A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication.
2004,
Pubmed
,
Xenbase
Shechter,
ATR and ATM regulate the timing of DNA replication origin firing.
2004,
Pubmed
,
Xenbase
Stokes,
DNA replication is required for the checkpoint response to damaged DNA in Xenopus egg extracts.
2002,
Pubmed
,
Xenbase
Tercero,
A central role for DNA replication forks in checkpoint activation and response.
2003,
Pubmed
Tutter,
Chromosomal DNA replication in a soluble cell-free system derived from Xenopus eggs.
2006,
Pubmed
,
Xenbase
Walter,
Regulated chromosomal DNA replication in the absence of a nucleus.
1998,
Pubmed
,
Xenbase
Walter,
Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase alpha.
2000,
Pubmed
,
Xenbase
Ward,
UV-induced ataxia-telangiectasia-mutated and Rad3-related (ATR) activation requires replication stress.
2004,
Pubmed
Yanow,
Xenopus Drf1, a regulator of Cdc7, displays checkpoint-dependent accumulation on chromatin during an S-phase arrest.
2003,
Pubmed
,
Xenbase
Zhao,
ATR-mediated checkpoint pathways regulate phosphorylation and activation of human Chk1.
2001,
Pubmed
Zhou,
The DNA damage response: putting checkpoints in perspective.
2000,
Pubmed
