Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork.
Eukaryotic cells regulate the progression and integrity of DNA replication forks to maintain genomic stability and couple DNA synthesis to other processes. The budding yeast proteins Mrc1 and Tof1 associate with the putative MCM-Cdc45 helicase and limit progression of the replisome when nucleotides are depleted, and the checkpoint kinases Mec1 and Rad53 stabilize such stalled forks and prevent disassembly of the replisome. Forks also pause transiently during unperturbed chromosome replication, at sites where nonnucleosomal proteins bind DNA tightly. We describe a method for inducing prolonged pausing of forks at protein barriers assembled at unique sites on a yeast chromosome, allowing us to examine for the first time the effects of pausing upon replisome integrity. We show that paused forks maintain an intact replisome that contains Mrc1, Tof1, MCM-Cdc45, GINS, and DNA polymerases alpha and epsilon and that recruits the Rrm3 helicase. Surprisingly, pausing does not require Mrc1, although Tof1 and Csm3 are both important. In addition, the integrity of the paused forks does not require Mec1, Rad53, or recombination. We also show that paused forks at analogous barriers in the rDNA are regulated similarly. These data indicate that paused and stalled eukaryotic replisomes resemble each other but are regulated differently.
Calzada
Arturo
A
Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, UK.
Hodgson
Ben
B
Kanemaki
Masato
M
Bueno
Avelino
A
Labib
Karim
K
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004271:DNA, Fungal / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D004718:Saccharomycetales / Q000235:genetics
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C105579:FOB1 protein, S cerevisiae; C441579:MRC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C459081:Rrm3 protein, S cerevisiae; D004265:DNA Helicases