Dataset Information

Xenopus laevis Cdc45-FLAG interactions on chromatin during DNA replication

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Xenopus Laevis (african Clawed Frog)

TISSUE(S): Cell Culture, Oocyte

SUBMITTER: Aga Gambus

LAB HEAD: Dr Aga Gambus

PROVIDER: PXD044422 | Pride | 2023-10-27

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	MSB66501ABand_01.raw	Raw
	MSB66501ABand_01__F200115_.mzid.gz	Mzid
	MSB66501ABand_01__F200115_.mzid_MSB66501ABand_01__F200115_.MGF	Mzid
	MSB66501ABand_02.raw	Raw
	MSB66501ABand_02__F200116_.mzid.gz	Mzid

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Publications

The structural mechanism of dimeric DONSON in replicative helicase activation.

Cvetkovic Milos A MA Passaretti Paolo P Butryn Agata A Reynolds-Winczura Alicja A Kingsley Georgia G Skagia Aggeliki A Fernandez-Cuesta Cyntia C Poovathumkadavil Divyasree D George Roger R Chauhan Anoop S AS Jhujh Satpal S SS Stewart Grant S GS Gambus Agnieszka A Costa Alessandro A

Molecular cell 20231010 22

The MCM motor of the replicative helicase is loaded onto origin DNA as an inactive double hexamer before replication initiation. Recruitment of activators GINS and Cdc45 upon S-phase transition promotes the assembly of two active CMG helicases. Although work with yeast established the mechanism for origin activation, how CMG is formed in higher eukaryotes is poorly understood. Metazoan Downstream neighbor of Son (DONSON) has recently been shown to deliver GINS to MCM during CMG assembly. What im ...[more]

PMID: 37820732

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