MCM2-7 Form Double Hexamers at Licensed Origins in Xenopus Egg Extract

Agnieszka Gambus,Guennadi A Khoudoli,Richard C Jones,J Julian Blow

doi:10.1074/jbc.m110.199521

Abstract

In late mitosis and G1, Mcm2-7 are assembled onto replication origins to license them for initiation in the upcoming S phase. After initiation, Mcm2-7 provide helicase activity to unwind DNA at the replication fork. Here we examine the structure of Mcm2-7 on chromatin in Xenopus egg extracts. We show that prior to replication initiation, Mcm2-7 is present at licensed replication origins in a complex with a molecular mass close to double that of the Mcm2-7 hexamer. This complex has approximately stoichiometric quantities of the 6 Mcm2-7 proteins and we conclude that it consists of a double heterohexamer. This provides a configuration potentially capable of initiating a pair of bidirectional replication forks in S phase. We also show that after initiation, Mcm2-7 associate with Cdc45 and GINS to form a relatively stable CMG (Cdc45-MCM-GINS) complex. The CMG proteins also associate less strongly with other replication proteins, consistent with the idea that a single CMG complex forms the core of the replisome.

Highlights

In eukaryotes, precise duplication of the genome during S phase is achieved through the initiation of replication forks at numerous origins distributed throughout the DNA
We provide evidence that during S phase in the Xenopus system, Mcm2-7 associate with Cdc45 and GINS to form CMG complexes only when replication forks have been initiated
Because most of the chromatin-bound Mcm2-7 license dormant replication origins that remain inactive during normal S phases [25,26,27] this result is consistent with the idea that Mcm2-7 on licensed origins are soluble in low salt, but become insoluble when they associate with replication fork proteins in the active replisome

Summary

EXPERIMENTAL PROCEDURES

Xenopus Egg Extract—Metaphase-arrested Xenopus laevis egg extracts were prepared as previously described [18]. Gel Filtration and Glycerol Gradients—Chromatin samples for gel filtration or glycerol gradient were prepared as described above; extract samples were prepared by adding 4 volumes of ANIB buffer ϩ10% sucrose and centrifuging 10 min, 20,000 ϫ g, 4 °C to remove insoluble material. After 5 ϫ 5 min washes with ANIB, beads were boiled in 70 ␮l of LDS sample buffer (Invitrogen) and 40-␮l IP samples were run on a 4 –12% gradient NuPAGE gel (Invitrogen). For Cdc and Ctf immunoprecipitation, chromatin was isolated in mid-S phase from a 4-ml reaction; 500 ␮l of antibody-coupled beads were used and 40-␮l IP samples run on 10% NuPAGE gel (Invitrogen) in MOPS buffer for 2 cm, lanes cut into 10 slices for analysis by mass spectrometry. Cipitated with methanol/chloroform, prepared for mass spectrometry and protein abundance was estimated from total ion current as described [21, 24]

RESULTS

C Identified Spectral Count Spectral Count protein control IP

C Identified protein

DISCUSSION