Michael Leffak (Advisor)
Master of Science (MS)
The DNA unwinding element binding protein (DUE-B) was first identified by using a yeast one hybrid screen with the DNA unwinding element (DUE) from the c-myc origin as bait. DUE-B's orthologue in the yeast Saccharomyces cerevisiae lacks the last 60 C-terminal amino acids and has been identified as a D-tyrosyl-tRNA deacylase. A substantial group of evidence suggests a role for DUE-B in the regulation of replication initiation. Here we show that DUE-B is focused in nuclear speckles and colocalizes with spliceosome associated protein 145 (SAP145), an mRNA splicing factor 3B subunit. Mass spectrometry results show that SAP145 co-purifies with the 6xHis-tagged DUE-B. Surprisingly, ÄCT- DUE-B, the DUE-B mutant lacking the 60 C-terminal amino acids, appeared almost exclusively in nuclear speckles whereas its yeast orthologue was found to be cytoplasmic. DUE-B's distribution pattern did not change in cells arrested in G1/S and it did not appear in replication foci despite the strong evidence of its involvement in replication initiation. Finally, DUE-B's proposed interaction with DNA methyltransferase 1 (Dnmt1) is further investigated by immunofluorescence. Interestingly, results show that this interaction with Dnmt1 seems to occur in nuclear speckles. Based on DUE-B's interacting proteins and its concentration in nuclear speckles, a possible role in the DNA damage response is discussed.
Department or Program
Department of Biochemistry and Molecular Biology
Year Degree Awarded
Copyright 2007, all rights reserved. This open access ETD is published by Wright State University and OhioLINK.