Dynamic association of orca with prereplicative complex components regulates dna replication initiation

Zhen Shen, Arindam Chakraborty, Ankur Jain, Sumanprava Giri, Taekjip Ha, Kannanganattu V. Prasanth, Supriya G. Prasanth

Research output: Contribution to journalArticle

Abstract

In eukaryotes, initiation of DNA replication requires the assembly of a multiprotein prereplicative complex (pre-RC) at the origins. We recently reported that aWDrepeat-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 G1-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 G1, and with geminin in post-G1 cells. Overexpression of geminin results in the loss of interaction between ORCA and Cdt1, suggesting that increased levels of geminin in post-G1 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.

Original languageEnglish (US)
Pages (from-to)3107-3120
Number of pages14
JournalMolecular and cellular biology
Volume32
Issue number15
DOIs
StatePublished - Aug 1 2012
Externally publishedYes

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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