Deletion of the cruciform binding domain in CBP/14-3-3 displays reduced origin binding and initiation of DNA replication in budding yeast.
BACKGROUND
Initiation of eukaryotic DNA replication involves many protein-protein and protein-DNA interactions. We have previously shown that 14-3-3 proteins bind cruciform DNA and associate with mammalian and yeast replication origins in a cell cycle dependent manner.


RESULTS
By expressing the human 14-3-3epsilon, as the sole member of 14-3-3 proteins family in Saccharomyces cerevisiae, we show that 14-3-3epsilon complements the S. cerevisiae Bmh1/Bmh2 double knockout, conserves its cruciform binding activity, and associates in vivo with the yeast replication origins ARS307. Deletion of the alpha5-helix, the potential cruciform binding domain of 14-3-3, decreased the cruciform binding activity of the protein as well as its association with the yeast replication origins ARS307 and ARS1. Furthermore, the mutant cells had a reduced ability to stably maintain plasmids bearing one or multiple origins.


CONCLUSIONS
14-3-3, a cruciform DNA binding protein, associates with yeast origins of replication and functions as an initiator of DNA replication, presumably through binding to cruciform DNA forming at yeast replicators.
Yahyaoui
Wafaa
W
McGill Cancer Centre, Montreal, Quebec, Canada. wafaa.yahyaoui@mcgill.ca <wafaa.yahyaoui@mcgill.ca>
Callejo
Mario
M
Price
Gerald B
GB
Zannis-Hadjopoulos
Maria
M
D048948:14-3-3 Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D045566:DNA, Cruciform / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D018741:Replication Origin / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion / Q000235:genetics*
D048948:14-3-3 Proteins; C075852:BMH1 protein, S cerevisiae; C097164:BMH2 protein, S cerevisiae; D002843:Chromatin; D045566:DNA, Cruciform; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins; C482983:YWHAE protein, human