Michael Jenkyn-Bedford / Morgan L Jones / Yasemin Baris / Karim P M Labib / Giuseppe Cannone / Joseph T P Yeeles / Tom D Deegan /
PubMed 要旨
Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase. Despite being driven by evolutionarily ...Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCF in budding yeast, CUL2 in metazoa), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA. However, it is unknown how SCF and CUL2 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome-E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR-MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity.
EMDB-13494, PDB-7plo: H. sapiens replisome-CUL2/LRR1 complex 手法: EM (単粒子) / 解像度: 2.8 Å
EMDB-13495: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, bound to dsDNA (conformation I) - full complex (unsharpened map) 手法: EM (単粒子) / 解像度: 3.9 Å
EMDB-13496: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, bound to dsDNA (conformation I) - full complex (sharpened map) 手法: EM (単粒子) / 解像度: 3.4 Å
EMDB-13497: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, bound to dsDNA (conformation I) - MCM N-tier region 手法: EM (単粒子) / 解像度: 3.3 Å
EMDB-13498: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, bound to dsDNA (conformation I) - MCM C-tier region 手法: EM (単粒子) / 解像度: 3.3 Å
EMDB-13500: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, bound to dsDNA (conformation II) - full complex (unsharpened map) 手法: EM (単粒子) / 解像度: 4.4 Å
EMDB-13512: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, bound to dsDNA (conformation II) - full complex (sharpened map) 手法: EM (単粒子) / 解像度: 3.7 Å
EMDB-13513: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, bound to dsDNA (conformation II) - MCM N-tier region 手法: EM (単粒子) / 解像度: 3.4 Å
EMDB-13514: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, bound to dsDNA (conformation II) - MCM C-tier region 手法: EM (単粒子) / 解像度: 3.6 Å
EMDB-13515: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, bound to dsDNA - region encompassing Dia2-Skp1 手法: EM (単粒子) / 解像度: 4.0 Å
EMDB-13516: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, bound to dsDNA - region encompassing Cdc45-GINS-Ctf4-Dpb2N 手法: EM (単粒子) / 解像度: 3.2 Å
EMDB-13517: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, bound to dsDNA - region encompassing Pol2-Dpb2-Mcm5 WH 手法: EM (単粒子) / 解像度: 3.5 Å
EMDB-13518: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, bound to dsDNA, enriched for particles containing Csm3-Tof1 手法: EM (単粒子) / 解像度: 3.6 Å
EMDB-13519: Cryo-EM reconstruction of the full S. cerevisiae replisome-SCF(Dia2) complex in the absence of DNA (unsharpened map) 手法: EM (単粒子) / 解像度: 8.3 Å
EMDB-13534: Cryo-EM map of the core human replisome on a DNA substrate lacking a 5'-flap 手法: EM (単粒子) / 解像度: 3.1 Å
EMDB-13539, PDB-7pmn: S. cerevisiae replisome-SCF(Dia2) complex bound to double-stranded DNA (conformation II) 手法: EM (単粒子) / 解像度: 3.2 Å
EMDB-13540: Cryo-EM reconstruction of the S. cerevisiae replisome-SCF(Dia2) complex, in the absence of DNA - region encompassing MCM N-tier-Cdc45-GINS-Ctf4-Dia2 手法: EM (単粒子) / 解像度: 3.9 Å