The DNA unwinding element binding protein DUE-B interacts with Cdc45 in preinitiation complex formation.
Template unwinding during DNA replication initiation requires the loading of the MCM helicase activator Cdc45 at replication origins. We show that Cdc45 interacts with the DNA unwinding element (DUE) binding protein DUE-B and that these proteins localize to the DUEs of active replication origins. DUE-B and Cdc45 are not bound at the inactive c-myc replicator in the absence of a functional DUE or at the recently identified ataxin 10 (ATX10) origin, which is silent before disease-related (ATTCT)(n) repeat length expansion of its DUE sequence, despite the presence of the origin recognition complex (ORC) and MCM proteins at these origins. Addition of a heterologous DUE to the ectopic c-myc origin, or expansion of the ATX10 DUE, leads to origin activation, DUE-B binding, and Cdc45 binding. DUE-B, Cdc45, and topoisomerase IIbeta binding protein 1 (TopBP1) form complexes in cell extracts and when expressed from baculovirus vectors. During replication in Xenopus egg extracts, DUE-B and Cdc45 bind to chromatin with similar kinetics, and DUE-B immunodepletion blocks replication and the loading of Cdc45 and a fraction of TopBP1. The coordinated binding of DUE-B and Cdc45 to origins and the physical interactions of DUE-B, Cdc45, and TopBP1 suggest that complexes of these proteins are necessary for replication initiation.
Chowdhury
A
A
Department of Biochemistry and Molecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio 45435, USA.
Liu
G
G
Kemp
M
M
Chen
X
X
Katrangi
N
N
Myers
S
S
Ghosh
M
M
Yao
J
J
Gao
Y
Y
Bubulya
P
P
Leffak
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D051738:Origin Recognition Complex / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D021381:Protein Transport; D018741:Replication Origin; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D014981:Xenopus
C113571:CDC45 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; C500359:DTD1 protein, human; D009687:Nuclear Proteins; C497224:ORC2 protein, human; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; C426020:Sld3 protein, S cerevisiae; C110513:TOPBP1 protein, human