Interaction between DUE-B and Treslin is required to load Cdc45 on chromatin in human cells

Sumeet Poudel,Jianhong Yao,Michael G Kemp,Michael Leffak

doi:10.1074/jbc.ra118.004519

Sumeet Poudel, Jianhong Yao + Show 2 more

Open Access

https://doi.org/10.1074/jbc.ra118.004519

Copy DOI

Abstract

A key step in the initiation of eukaryotic DNA replication is the binding of the activator protein Cdc45 to promote MCM helicase unwinding of the origin template. We show here that the c-myc origin DNA unwinding element-binding protein, DUE-B, interacts in HeLa cells with the replication initiation protein Treslin to allow Cdc45 loading onto chromatin. The chromatin loading of DUE-B and Treslin are mutually dependent, and the DUE-B-Treslin interaction is cell cycle-regulated to peak as cells exit G1 phase prior to the initiation of replication. The conserved C-terminal domain of DUE-B is required for its binding to TopBP1, Treslin, Cdc45, and the MCM2-7 complex, as well as for the efficient loading of Treslin, Cdc45, and TopBP1 on chromatin. These results suggest that DUE-B acts to identify origins by MCM binding and serves as a node for replication protein recruitment and Cdc45 transfer to the prereplication complex.

Highlights

A key step in the initiation of eukaryotic DNA replication is the binding of the activator protein Cdc[45] to promote MCM helicase unwinding of the origin template
DNA-unwinding element (DUE)-B and TopBP1 bound with similar kinetics slightly before Treslin and substantially before Cdc[45] loading during G1/S (T ϭ 9 –12 h)
These results are consistent with previous observations that DUE-B binds to sperm chromatin or plasmid DNA before Cdc[45] or RPA in Xenopus egg extracts and that DUE-B immunodepletion blocks subsequent Cdc[45] and RPA loading onto sperm chromatin (4)

Summary

Results

DUE-B binding to sperm chromatin in Xenopus egg extracts is preceded by TopBP1 binding and followed by Cdc[45] binding (4). DUE-B and TopBP1 bound with similar kinetics slightly before Treslin and substantially before Cdc[45] loading during G1/S (T ϭ 9 –12 h) These results are consistent with previous observations that DUE-B binds to sperm chromatin or plasmid DNA before Cdc[45] or RPA in Xenopus egg extracts and that DUE-B immunodepletion blocks subsequent Cdc[45] and RPA loading onto sperm chromatin (4). The C-terminal domain of DUE-B is essential for Cdc[45] loading in Xenopus egg extracts and onto HeLa chromatin (5), whereas the C terminus deletion mutant of DUE-B (⌬CT DUE-B) does not bind to Cdc[45] or TopBP1 (4). Considered together, these observations suggested that the C terminus of DUE-B may be required for the chromatin loading of Treslin and for the Treslin–DUE-B interaction. ⌬CT DUE-B was deficient in co-immunoprecipitation of Treslin (Fig. 6B), and Treslin could not pull down ⌬CT DUE-B (Fig. 6C)

26 His6-DUE-B

Discussion

Experimental procedures