Dynamic association of ORCA with prereplicative complex components regulates DNA replication initiation.
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.
Department of Cell and Developmental Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Zhen
Z
Shen
Arindam
A
Chakraborty
Ankur
A
Jain
Sumanprava
S
Giri
Taekjip
T
Ha
Kannanganattu V
KV
Prasanth
Supriya G
SG
Prasanth
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D059585:G1 Phase Cell Cycle Checkpoints; D064248:Geminin; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D000071456:Sequestosome-1 Protein
D048868:Adaptor Proteins, Signal Transducing; C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C577995:GMNN protein, human; D064248:Geminin; C497224:ORC2 protein, human; D051738:Origin Recognition Complex; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein