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

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.

PHIP as a therapeutic target for driver-negative subtypes of melanoma, breast, and lung cancer

“…Our results are supported by a recent chromatin proteomic analysis that identified an association of PHIP with histone-marked genomic regions (28). In addition, an important role for PHIP in regulating site-specific initiation of DNA replication and cell cycle progression has been recently reported (11). DNA synthesis and histone modifications are highly coordinated to ensure the uninterrupted advance of the replication fork and assembly of the nascent DNA strands onto nucleosomes, a process that involves the deposition of histones H3 and H4 (29,30).…”

“…S2 A-C). In addition, as a recent study has shown an important role for PHIP in DNA replication(11), we observed significant overexpression of the proliferation markers PCNA, Ki67, and phospho-Histone H3 (pHH3) on PHIP overexpression(Fig. 2F and G and SI Appendix, Fig.…”

The RepID–CRL4 ubiquitin ligase complex regulates metaphase to anaphase transition via BUB3 degradation

“…To determine the chromatin-association dynamics of RepID during the cell cycle, we have arrested HCT116 cells in early mitosis by nocodazole, then released the cells into nocodazole-free medium and analyzed cell cycle progression. Surprisingly, we noticed that RepIDdeficient (RepID knockout (KO)) cells 13 were significantly delayed in exiting mitosis and entering G1 phase as compared to RepID-expressing (RepID wild type (WT)) cells (Fig. 1b, c and Supplementary Fig.…”

“…First, DCAFs that contain a proliferating cell nuclear antigen (PCNA) interaction (PIP) domain, such as CDT2, recruit CRL4 to chromatin by associating with PCNA during DNA synthesis 10,11 . Second, another DCAF, replication initiation determinant protein (RepID)/PHIP/DCAF14, which contains a bromodomain 9 and a cryptic Tudor domain 12 , can bind replication origins 13 and recruit CRL4 to chromatin prior to DNA replication 14 . During the G1-S transition of the cell cycle, CRL4 ubiquitinates CDT1, a licensing factor and member of the pre-replication complex (Fig.…”

“…Surprisingly, we find that CRL4, which primarily is thought to regulate DNA replication and repair, plays a crucial role during mitosis by facilitating the ubiquitination of the SAC component BUB3, leading to the inactivation of the SAC and to the subsequent activation of APC/C and exit from mitosis. CRL4 is recruited to chromatin by the replication origin binding protein and metastatic melanoma marker RepID (DCAF14/PHIP) 13,34 . We find that, during mitosis, chromatin-bound CRL4 dissociates from RepID and binds another DCAF, tubulin-associated retinoblastoma binding protein 7 (RBBP7), which acts as a substrate receptor for BUB3.…”