PXD044422

PXD044422 is an original dataset announced via ProteomeXchange.

Title	Xenopus laevis Cdc45-FLAG interactions on chromatin during DNA replication
Description	The aim of the project was to purify Xenopus replisome from replicationg chromatin assembled in Xenopus laevis egg extract. To this end recombinant Cdc45-TEV-His10-FLAG5 was expressed in bacteria and purified. 4 ml of Xenopus laevis egg extract was activated into interphase and supplemented with 10 ng/µl of demembranated sperm DNA, 70 nM recombinant Cdc45, 40 µM aphidicolin, 5 mM caffeine and incubated at 23°C for 60 min. Chromatin was isolated in ANIB100 buffer (50 mM HEPES pH 7.6, 100 mM KOAc, 10 mM MgOAc, 2.5 mM Mg-ATP, 0.5 mM spermidine, 0.3 mM spermine, 1 µg/ml of each aprotinin, leupeptin and pepstatin, 25 mM β-glycerophosphate, 0.1 mM Na3VO4 and 10 mM 2-chloroacetamide) as described previously (Gillespie, Gambus et al. 2012). Chromatin pellets re-suspended in ANIB100 containing 20% sucrose. Protein complexes were released from chromatin by digestion with 4 U/µl benzonase nuclease (E1014-25KU, Sigma) and sonicated for 5 min using a sonicator with settings: 30 sec on, 30 sec off, low power (Bioruptor Diagenode UCD-200). Immunoprecipitation was performed using 100 µl of anti-FLAG M2 magnetic beads (Sigma-Aldrich). Before elution the sample was washed four times with 1 ml of ANIB100 20% sucrose, ANIB100 20% sucrose 0.1% Triton X-100, ANIB100 and elution buffer (25 mM HEPES pH 7.5, 100 mM KAc, 5 mM MgAc, 1 mM ATP and 0.02% NP-40), respectively. The sample was eluted adding 250 µM 3xFLAG peptide (Stratech) to 200 µl of elution buffer and a small proportion of it analysed by mass spectrometry.
HostingRepository	PRIDE
AnnounceDate	2023-11-14
AnnouncementXML	Submission_2023-11-14_09:13:40.541.xml
DigitalObjectIdentifier	https://dx.doi.org/10.6019/PXD044422
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Supported dataset by repository
PrimarySubmitter	Aga Gambus
SpeciesList	scientific name: Xenopus laevis (African clawed frog); NCBI TaxID: 8355;
ModificationList	phosphorylated residue; monohydroxylated residue; deamidated residue; iodoacetamide derivatized residue
Instrument	Orbitrap Fusion Lumos

Revision	Datetime	Status	ChangeLog Entry
0	2023-08-07 13:16:26	ID requested
1	2023-10-27 02:32:31	announced
⏵ 2	2023-11-14 09:13:46	announced	2023-11-14: Updated project metadata.

10.6019/PXD044422;

Cvetkovic MA, Passaretti P, Butryn A, Reynolds-Winczura A, Kingsley G, Skagia A, Fernandez-Cuesta C, Poovathumkadavil D, George R, Chauhan AS, Jhujh SS, Stewart GS, Gambus A, Costa A, The structural mechanism of dimeric DONSON in replicative helicase activation. Mol Cell, 83(22):4017-4031.e9(2023) [pubmed]

submitter keyword: DNA replication, Xenopus laevis, Cdc45, replisome

Dr Aga Gambus
contact affiliation	Chromosomal Replication Laboratory, Institute for Cancer and Genomic Sciences, IBR west, WX2.62, University of Birmingham, Edgbaston, Birmingham, B15 2TT
contact email	a.gambus@bham.ac.uk
lab head
Aga Gambus
contact affiliation	University of Birmingham
contact email	a.gambus@bham.ac.uk
dataset submitter

Dataset FTP location NOTE: Most web browsers have now discontinued native support for FTP access within the browser window. But you can usually install another FTP app (we recommend FileZilla) and configure your browser to launch the external application when you click on this FTP link. Or otherwise, launch an app that supports FTP (like FileZilla) and use this address: ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2023/10/PXD044422

PRIDE project URI

• PRIDE
  1. PXD044422
     1. Label: PRIDE project
     2. Name: Xenopus laevis Cdc45-FLAG interactions on chromatin during DNA replication