A replicator-specific binding protein essential for site-specific initiation of DNA replication in mammalian cells.

Ya Zhang,William C Reinhold,Michael Ryan,Chii Mei Lin,Liang Huang,Harlington Hanna,Mishal Rao,Ryangguk Kim,Christophe E Redon,Yang You,Owen K Smith,Qiaoming Long,Yves Boisclair,Koichi Utani,Haiqing Fu,Mirit I Aladjem

doi:10.1038/ncomms11748

Abstract

Mammalian chromosome replication starts from distinct sites; however, the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding. Here we identify a replication-initiation determinant (RepID) protein that binds a subset of replication-initiation sites. A large fraction of RepID-binding sites share a common G-rich motif and exhibit elevated replication initiation. RepID is required for initiation of DNA replication from RepID-bound replication origins, including the origin at the human beta-globin (HBB) locus. At HBB, RepID is involved in an interaction between the replication origin (Rep-P) and the locus control region. RepID-depleted murine embryonic fibroblasts exhibit abnormal replication fork progression and fewer replication-initiation events. These observations are consistent with a model, suggesting that RepID facilitates replication initiation at a distinct group of human replication origins.

Highlights

Mammalian chromosome replication starts from distinct sites; the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding
We found that 82.3% of replication-initiation determinant (RepID)-binding sites localized within 2 kb of replication-initiation sites, whereas 15.4% of replication-initiation sites localized within 2 kb of a RepIDbound region. (The cutoff at 2 kb was based on the size of the isolated nascent strands, which ranged between 0.5 and 1 kb as described in the Methods section.) When this analysis was expanded to consider a 5-kb distance, 86% of RepID-bound regions (20,841 of the total 24,222 sites) colocalized with replication-initiation events and 20.7% of replication-initiation sites colocalized with RepID-bound regions
In this study, we report that the RepID protein binds distinct mammalian replication origins and is required for sequencespecific initiation of DNA replication at these origins

Summary

Introduction

Mammalian chromosome replication starts from distinct sites; the principles governing initiation site selection are unclear because proteins essential for DNA replication do not exhibit sequence-specific DNA binding. No sequence-specific DNA-binding proteins were yet shown to determine replicator-specific initiation in mammalian cells, these studies indicate that distinct proteins might interact with subsets of origins, and recruit the general replication machinery to those sites. The human beta-globin locus (HBB) contains two intensely studied replicators residing in the replication-initiation regions (IRs)[31,32,33,34,35] This IR is used in both erythroid and non-erythroid cells, but initiates DNA replication during early stages of the S phase in erythroid cells and later during the S phase in non-erythroid cells[11,36,37,38,39,40]. The HBB IR, provides an excellent system to study replicatorbinding proteins as well as an opportunity to study replication timing

Methods

Results

Conclusion