SINGLE MOLECULE ANALYSIS OF COMPLEX DNA METABOLISM

University Of Texas Hlth Sci Ctr San Ant, San Antonio TX

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Abstract

Description: (Verbatim from the applicant's abstract) Metabolism-driven change in the conformation of double-stranded DNA is studied to understand several biological events. These events include the packaging of a double-stranded DNA genome in the preformed protein container (capsid) of a bacteriophage. The DNA packaging substrate for some bacteriophages is an end-to-end multimer (concatemer) of the mature genome. The concatemer is cleaved to genomes during packaging. Understanding of these metabolic changes has not been achieved conventionally. One reason is that any given macroscopically-observed change in DNA conformation is unsynchronized and, possibly, variable among microscopically-observed single DNA molecules. Another reason is that these changes occur in complex systems that may consist of more than one phase. The limitations of conventional procedures can be bypassed by using single molecule-fluorescence microscopy. Metabolic events are imaged directly at a resolution of approximately 250 nm. Higher resolution information is obtained by more indirect use of the data from fluorescence microscopy. The PI's laboratory has recently solved the problems that had previously prevented use of this strategy. Both an initiating cleavage of concatemers and entry of DNA into capsids have been observed by singe-molecule fluorescence microscopy. The following are the specific aims of the current proposal: (1) The behavior of single capsids will be observed by fluorescence microscopy before the initiation of packaging of concatemer-associated genomes of bacteriophage T7. Determination will be made of whether capsids search for initiation sites by use of an ATP-driven motor. (2) The formation of the initiation complex will be observed by single-molecule fluorescence microscopy. A hypothesis that proposes initiation via capsid-induced cyclization will be tested. (3) The entry of DNA into a capsid will be observed while the capsid is attached to a solid support. Hypotheses for the mechanism of pumping DNA into a capsid will undergo preliminary tests. (4) Procedures will be further developed for the purpose of analyzing DNA metabolism by single-molecule fluorescence microscopy. The research proposed constitutes a new strategy for understanding the biochemistry of complex systems. This research also reveals the biological and physical constraints that were present when these systems evolved. The procedures and concepts developed will assist in the analysis of all biological systems. They also have use in both environmental biology and disease diagnosis.