RecQ4 promotes the conversion of the pre-initiation complex at a site-specific origin for DNA unwinding inXenopusegg extracts

Abstract

Eukaryotic DNA replication is initiated through stepwise assembly of evolutionarily conserved replication proteins onto replication origins, but how the origin DNA is unwound during the assembly process remains elusive. Here, we established a site-specific origin on a plasmid DNA, using in vitro replication systems derived from Xenopus egg extracts. We found that the pre-replicative complex (pre-RC) was preferentially assembled in the vicinity of GAL4 DNA-binding sites of the plasmid, depending on the binding of Cdc6 fused with a GAL4 DNA-binding domain in Cdc6-depleted extracts. Subsequent addition of nucleoplasmic S-phase extracts to the GAL4-dependent pre-RC promoted initiation of DNA replication from the origin, and components of the pre-initiation complex (pre-IC) and the replisome were recruited to the origin concomitant with origin unwinding. In this replication system, RecQ4 is dispensable for both recruitment of Cdc45 onto the origin and stable binding of Cdc45 and GINS to the pre-RC assembled plasmid. However, both origin binding of DNA polymerase a and unwinding of DNA were diminished upon depletion of RecQ4 from the extracts. These results suggest that RecQ4 plays an important role in the conversion of pre-ICs into active replisomes requiring the unwinding of origin DNA in vertebrates.

Using Xenopus to understand human disease and developmental disorders

“…The pursuit of new discoveries regarding fundamental biological processes is still a major rationale for using Xenopus as a model system. For example, investigations of the molecular regulation of the cell cycle continue to use Xenopus oocytes, eggs and embryos as a major model (Malhotra, Vinod, Mansfeld, Stemmann, & Mayer, ; Sabherwal, Thuret, Lea, Stanley, & Papalopulu, ; Sanuki et al, ). Xenopus also has greatly enhanced our understanding of DNA replication and repair (Kalb, Mallery, Larkin, Huang, & Hiom, ; Long, Joukov, Budzowska, & Walter, ; Räschle et al, ; Shintomi, Takahashi, & Hirano, ; Zhang et al, ), and the mechanisms of genome organization, transcriptional regulation, and epigenetics (Belikov, Berg, & Wrange, ; Bogdanović et al, ; Buisine et al, ; Gazdag, Jacobi, van Kruijsbergen, Weeks, & Veenstra, ; Gao et al, ; Hontelez et al, ; Owens et al, ; Tamaoki et al, ; Wang et al, ; Wen, Fu, Guo, Chen, & Shi, ).…”

Suppression of targeting of Dbf4‐dependent kinase to pre‐replicative complex in G0 nuclei

“…Plasmid DNA was prepared and attached to superparamagnetic beads as described previously (Sanuki et al., ; Waga & Zembutsu, ). To assemble pre‐RC on plasmid DNA, 100 ng of template DNA was suspended in 100 μl of ice‐cold EB supplemented with 0.25% NP40.…”

MCM interference during licensing of DNA replication inXenopusegg extracts-Possible Role of a C-terminal region of MCM3-

“…Xenopus egg extracts and sperm chromatin were prepared as described, immunodepletion was performed as described [41,48]. To prepare heat sup, we incubated egg extracts at 988C for 2 min, and then centrifuged them at 15000 rpm for 15 min at 48C.…”

“…DNA-coupled beads were prepared as described [48]. Briefly, biotinylated plasmids obtained by UV cross-linking were incubated with streptavidin-coupled Dynabeads (Thermo Fisher Scientific).…”