Structure and Function of the c-myc DNA-unwinding Element-binding Protein DUE-B

Abstract

Local zones of easily unwound DNA are characteristic of prokaryotic and eukaryotic replication origins. The DNA-unwinding element of the human c-myc replication origin is essential for replicator activity and is a target of the DNAunwinding element-binding protein DUE-B in vivo. We present here the 2.0 Å crystal structure of DUE-B and complementary biochemical characterization of its biological activity. The structure corresponds to a dimer of the N-terminal domain of the full-length protein and contains many of the structural elements of the nucleotide binding fold. A single magnesium ion resides in the putative active site cavity, which could serve to facilitate ATP hydrolytic activity of this protein. The structure also demonstrates a notable similarity to those of tRNA-editing enzymes. Consistent with this structural homology, the N-terminal core of DUE-B is shown to display both D-aminoacyl-tRNA deacylase activity and ATPase activity. We further demonstrate that the C-terminal portion of the enzyme is disordered and not essential for dimerization. However, this region is essential for DNA binding in vitro and becomes ordered in the presence of DNA.During each division of somatic cells DNA replication is regulated so that the genome is copied in its entirety only once (1, 2). The classical replicon hypothesis states that the initiation of DNA replication at replication origins is controlled by initiator protein binding at regulatory sites called replicators and has served as a useful description for prokaryotic replication (3). In contrast, a concise description of eukaryotic replication is complicated by the fact that eukaryotic chromosomes contain multiple origins of replication and the replicator sequences controlling them do not share easily recognizable conserved sequences (4, 5). However, a structure common to yeast and mammalian origins of replication is a region of easily unwound DNA termed a DNAunwinding element (DUE) 3 (6 -13). Replication initiates within a 3.5-kb region upstream of the human c-myc gene (14 -16). Semi-conservative DNA synthesis was shown to originate in this region using nascent DNA abundance assays in chromosomal and plasmid-based systems in vitro and in vivo (13, 16 -26). Transposition of a 2.4-kb c-myc origin fragment to an ectopic site in the HeLa genome promotes bidirectional DNA replication initiation at the site of insertion, confirming its function as a chromosomal replicator (13,22). This 2.4-kb region contains an AT-rich region comprising three matches to the Saccharomyces cerevisiae ARS consensus sequence and a ϳ40-bp DUE zone of predicted helical instability, also termed the far upstream element or FUSE (27), which has been shown to be sensitive to single stranddirected reagents in vitro and in vivo (19,27). Deletion of the DUE/ARS region eliminates c-myc origin activity (13), and a heterologous DUE restores origin activity, 4 implying that a DUE is essential for chromosomal replication origin activity.In addition to a region of easily unwound DNA, replication origins ...

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

“…Like Y RNAs, DUE-B is released from the DNA template during initiation (Chowdhury et al, 2010). Interestingly, DUE-B has structural similarity to aminoacyl-tRNA editing enzymes (Casper et al, 2005;Kemp et al, 2007), which might account for its molecular interaction with the highly structured hY RNAs. As with depletion of Y RNAs (Christov et al, 2006;Christov et al, 2008;Gardiner et al, 2009), depletion of DUE-B leads to an inhibition of DNA replication in cell-based and cell-free assays (Casper et al, 2005).…”

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

“…The conformation of the amino-terminal 150-residue domain of DUE-B shows strong evolutionary conservation to the structures of orthologous proteins in yeast, eubacteria, and archaebacteria (18). Remarkably, the structurally similar proteins in lower organisms do not appear to play direct roles in DNA replication but are involved in the proofreading of tRNA aminoacylation.…”

“…The 209-amino-acid (23.4-kDa) protein has been strongly conserved during metazoan evolution (18). By chromatin immunoprecipitation (ChIP), DUE-B has been shown to localize with MCM proteins to the DUEs of the c-myc and lamin B2 replication origins, and DUE-B binding to an ectopic copy of the c-myc core replicator is eliminated by deletion of the DUE (12).…”

“…Suggestive of an important role for DUE-B in DNA replication, DUE-B small interfering RNA (siRNA) slows the G 1 -to S-phase transition of HeLa cells. In the Xenopus egg extract cell-free replication system, DUE-B depletion inhibits DNA replication, and a dominant-negative form of DUE-B blocks replication after pre-RC formation but before origin unwinding and RPA binding (7,18).…”

“…Remarkably, the structurally similar proteins in lower organisms do not appear to play direct roles in DNA replication but are involved in the proofreading of tRNA aminoacylation. Indeed, human DUE-B retains the amino acid deacylase and esterase activities of these proteins (18). Carboxyl to the protease-resistant amino-terminal domain, human DUE-B also contains a 59-amino-acid extension not found in the tRNA deacylases.…”