A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase α within the eukaryotic replisome

Abstract

The eukaryotic replisome is a crucial determinant of genome stability, but its structure is still poorly understood. We found previously that many regulatory proteins assemble around the MCM2-7 helicase at yeast replication forks to form the replisome progression complex (RPC), which might link MCM2-7 to other replisome components. Here, we show that the RPC associates with DNA polymerase a that primes each Okazaki fragment during lagging strand synthesis. Our data indicate that a complex of the GINS and Ctf4 components of the RPC is crucial to couple MCM2-7 to DNA polymerase a. Others have found recently that the Mrc1 subunit of RPCs binds DNA polymerase epsilon, which synthesises the leading strand at DNA replication forks. We show that cells lacking both Ctf4 and Mrc1 experience chronic activation of the DNA damage checkpoint during chromosome replication and do not complete the cell cycle. These findings indicate that coupling MCM2-7 to replicative polymerases is an important feature of the regulation of chromosome replication in eukaryotes, and highlight a key role for Ctf4 in this process.

Interaction between human Ctf4 and the Cdc45/Mcm2-7/GINS (CMG) replicative helicase

“…S5C, Right)]. These results indicate that hCtf4 forms a complex with hGINS, as reported previously in yeast (22), but the human complex is less stable than the yeast Ctf4-GINS complex.…”

“…Weaker hCMG binding was detected with the N (WD + SepB) domains, whereas the WD, SepB, or HMG domain alone failed to bind the hCMG complex. Previous reports indicated that the budding yeast SepB domain alone mediated an interaction between Ctf4 and GINS (22), suggesting that the HMG domain of higher eukaryotic Ctf4 may have additional binding sites in the CMG complex important for the stability of the hCtf4-CMG complex.…”

“…Immunodepletion of Ctf4 in Xenopus egg extracts, as well as siRNA targeted destruction of human Ctf4 (hCtf4) in HeLa cells, markedly decreased DNA replication (7,19). Ctf4 was reported to interact with various replication proteins, including Mcm10, GINS, Pol α, Pol δ, and Pol e, and was reported to contribute to the stability of the p180 catalytic subunit of Pol α (7,(19)(20)(21)(22). Recently, Ctf4 was found to connect Pol α to the budding yeast RPC (21,22).…”

“…Ctf4 was reported to interact with various replication proteins, including Mcm10, GINS, Pol α, Pol δ, and Pol e, and was reported to contribute to the stability of the p180 catalytic subunit of Pol α (7,(19)(20)(21)(22). Recently, Ctf4 was found to connect Pol α to the budding yeast RPC (21,22). The association of Ctf4 with the RPC was shown to be dependent on the interaction of Ctf4 with GINS, a component of the CMG complex present in the RPC, suggesting a possible scaffolding function that coordinates the action of the replicative helicase and Pol α during DNA replication.…”

Structures of human primase reveal design of nucleotide elongation site and mode of Pol α tethering

“…On its turn, the Pol α/primase complex is anchored to the CMG helicase by the conserved Ctf4 replication factor, which acts as a bridge between the GINS component of the helicase and the catalytic subunit of Pol α (22,23). Thus, a chain of specific protein-protein interfaces links unwinding of the parental strands to priming of nucleic acid synthesis on template DNA.…”