Dynamic Association of ORCA with Prereplicative Complex Components Regulates DNA Replication Initiation

Abstract

In eukaryotes, initiation of DNA replication requires the assembly of a multiprotein prereplicative complex (pre-RC) at the origins. We recently reported that a WD repeat-containing protein, origin recognition complex (ORC)-associated (ORCA/LRWD1), plays a crucial role in stabilizing ORC to chromatin. Here, we find that ORCA is required for the G 1 -to-S-phase transition in human cells. In addition to binding to ORC, ORCA associates with Cdt1 and its inhibitor, geminin. Single-molecule pulldown experiments demonstrate that each molecule of ORCA can bind to one molecule of ORC, one molecule of Cdt1, and two molecules of geminin. Further, ORCA directly interacts with the N terminus of Orc2, and the stability of ORCA is dependent on its association with Orc2. ORCA associates with Orc2 throughout the cell cycle, with Cdt1 during mitosis and G 1 , and with geminin in post-G 1 cells. Overexpression of geminin results in the loss of interaction between ORCA and Cdt1, suggesting that increased levels of geminin in post-G 1 cells titrate Cdt1 away from ORCA. We propose that the dynamic association of ORCA with pre-RC components modulates the assembly of its interacting partners on chromatin and facilitates DNA replication initiation.

Orc2 protects ORCA from ubiquitin-mediated degradation

“…29 ORCA binds to Cdt1 and Geminin, and the timely association of ORCA with ORC, Cdt1 and Geminin and the binding of each of these components to chromatin dictated by ORCA ensures "once per cell cycle" replication. 58 In the present study, we demonstrate that ORCA is polyubiquitinated in vivo with elevated ubiquitination and degradation at the G 1 /S boundary and is stabilized when cells are treated with the proteasome inhibitor MG132. Orc2, the direct binding partner of ORCA, stabilizes ORCA by preventing its proteasomal degradation.…”

“…We have recently demonstrated that ORCA is required for ORC binding to chromatin. 29 Similarly, Orc2 is required for the cellular stability of ORCA 57,58 and Orc3 ( Fig. 5A and B).…”

Identification and characterization of human testis derived circular RNAs and their existence in seminal plasma

“…The seven host genes of ten testis specific circRNAs are LRWD1, STK31, HIST1H2BA, RNF17, BRDT, SMC1B and SPATA16 . In GO analysis, these genes are closely related to DNA replication33, spermatogonia development34, histone displacement35, androgen receptor interaction36, meiotic cell cycle37, male meiosis38 and spermatogenesis39 respectively.…”

Bringing single-molecule spectroscopy to macromolecular protein complexes

“…The researchers have also reported proof-of-principle studies using systems such as mTOR (mammalian target of rapamycin) protein complexes and membrane receptor proteins [17]. The same group has extended its research to stoichiometry analyses of AKAPs (A-kinase anchoring proteins) [48], ORCA (origin-recognition complex-associated) protein complexes [49], and peptide loading complexes [50].…”

“…The researchers have also reported proof-of-principle studies using systems such as mTOR (mammalian target of rapamycin) protein complexes and membrane receptor proteins [17]. The same group has extended its research to stoichiometry analyses of AKAPs (A-kinase anchoring proteins) [48], ORCA (origin-recognition complex-associated) protein complexes [49], and peptide loading complexes [50].Jain et al also demonstrated the analysis of endogenous proteins obtained from mouse brain and heart tissues ( Figure 4c). Although it is often demanding to analyze endogenous proteins due to their low abundance, this difficulty can be overcome by the high sensitivity of singlemolecule complex analysis.…”