Molecular analysis of the MLL breakpoint region in leukemia: a study of binding of DNA-topoisomerase II and/or other nuclear proteins to specific DNA breakpoint fragments.
The restricted size of the MLL breakpoint region, only 8kb, suggests that the mechanism of the translocations involve a specific DNA sequence(s) in the region. Several studies have suggested that the MLL breakpoint region is prone to cleavage by topo II, but the region has not yet been analyzed for direct binding of topo II or other nuclear proteins. We have used a standard gel shift mobility assay to determine if topo II protein and/or nuclear proteins normally bind to the MLL breakpoint region. Using Polymerase Chain Reaction (PCR) we amplified three 200 base pair DNA fragments from the breakpoint region. Two of the fragments, probes A and C, lie at the extreme 5' and 3' ends of the breakpoint region, respectively. Our third probe, B, is located at a proposed cleavage site for topo II in the MLL gene. Probes A, B, and C were labeled for chemiluminescent detection and then incubated with nuclear proteins from a human leukemia cell line, REH, in separate experiments. The binding of nuclear proteins to the probes was determined by retarded migration (shifting) of probes during polyacrylamide gel electrophoresis.
Our analysis indicates that probe A specifically binds one or more nuclear proteins from the REH cell line. In a modified "supershift" assay we tested the binding of the topo II antibody by adding a polyclonal antibody to topo II to the reaction. We preliminarily noted additional shifting of the probe, suggesting that the protein topo II is binding to probe A. Since topo II antibody binding was inconclusive and suitable antibody controls were not available, we took a more direct approach of using the purified topo II protein in the electrophoretic mobility shift assay. No shifting was detected using probe A and purified topo II, indicating a different nuclear protein is binding this region of the MLL breakpoint. From previous studies we had predicted that probe B would possibly bind topo II directly, but after many trials we did not detect binding of REH nuclear proteins to either probes B or C.
The correlation of topo II binding and MLL translocations is still a possibility. There are many more regions of the 8kb MLL breakpoint region yet to be analyzed for binding of topo II and/or nuclear proteins. Future studies will also focus on identification of the nuclear protein binding probe A.
Jessica D. Hamilton * and Heidi J. Super
Department of Biology, Minot State University, Minot, ND 58707
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|Title Annotation:||A. Rodger Denison Student Research Competition: COMMUNICATIONS: UNDERGRADUATE DIVISION|
|Author:||Hamilton, Jessica D.; Super, Heidi J.|
|Publication:||Proceedings of the North Dakota Academy of Science|
|Date:||Apr 1, 2004|
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