The DNA Unwinding Element Binding Protein DUE-B Interacts with Cdc45 in Preinitiation Complex Formation

Abstract

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.

Replication Fork Stalling and Checkpoint Activation by a PKD1 Locus Mirror Repeat Polypurine-Polypyrimidine (Pu-Py) Tract

“…Cells were grown at 37°C and 5% CO 2 in DMEM supplemented with 10% newborn calf serum. Nascent DNA was isolated from human cell lines by denaturing gel electrophoresis and quantitated by real time qPCR (38). qPCR results are based on at least two biological replicates (independent nascent DNA isolations) quantitated in triplicate.…”

“…The c-myc core replicator integrated at this ectopic site displays the replication timing, chromatin structure, and replication protein (origin recognition complex (ORC), Cdc6, minichromosome maintenance (MCM), DUE-B, and Cdc45) binding of the endogenous c-myc origin (38,51,52). Clonal c-myc:Pu-Py cell lines were created in which the Py-rich sequence is replicated as the leading or lagging strand template from the neighboring c-myc origin to determine whether alternative DNA structure formation results in an orientation-dependent barrier to replication fork movement.…”

Helicase Activation and Establishment of Replication Forks at Chromosomal Origins of Replication

“…Two additional proteins, GEMC1 (Balestrini et al 2010) and DUE-B (Chowdhury et al 2010), that bind to both TopBP1 and Cdc45 are required for the recruitment of Cdc45 to chromatin. However, their actual functions have not been elucidated.…”

Dynamic interaction of Y RNAs with chromatin and initiation proteins during human DNA replication

“…The DUE-B protein binds to DNA unwinding elements (DUEs) at human replication origins and is implicated in the unwinding step of origin activation (Casper et al, 2005;Chowdhury et al, 2010). DUE-B binding to origins is dependent on ORC, and is in turn required for the loading of CDC45 (Chowdhury et al, 2010).…”

“…Finally, DNA primase and polymerases are recruited to begin DNA synthesis, marking entry into S phase. Additional levels of control have evolved in metazoan organisms (Arias and Walter, 2007), and proteins such as DUE-B, high mobility group A (HMGA)1a and the Ku autoantigen have been implicated in the initiation of DNA replication in mammalian cells (Norseen et al, 2008;Rampakakis et al, 2008;Thomae et al, 2008;Chowdhury et al, 2010).…”