Single Molecule Studies of DNA 'Breathing' Fluctuations and their Roles in Helica

University Of Oregon, Eugene OR

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Abstract

DESCRIPTION (provided by applicant): The ability of the cellular machinery to accurately replicate and transcribe DNA is crucial to the healthy function of living organisms. While the components involved in these processes are largely known, the molecular mechanisms by which the components function are not well understood and, as a consequence, processes that result in malfunctions of the replication or transcription machinery are also poorly understood. Many diseases are a direct result of such malfunctions, including certain forms of cancer, and thus a better understanding of how these systems work may ultimately lead to cures for diseases which have heretofore remained elusive. Doubled-stranded (ds) DNA 'breathing' is a phenomenon that refers to the transient breaking of hydrogen-bonds and/or the unstacking of adjacent nucleic acid bases in dsDNA as a consequence of thermal fluctuations. The DNA breathing motions that result from these fluctuations are likely to be important components of the recognition mechanisms of specific base sequences by genome-regulatory proteins, including the replication helicases that catalyze the unwinding of double-stranded DNA during the processes of DNA replication and transcription. In the studies proposed we plan to use new single molecule spectroscopic approaches to observe and characterize the longer-lived (micro- to milliseconds) thermal fluctuations of DNA bases during the DNA breathing process. We will utilize visible wavelength fluorescent probes rigidly attached to double-stranded DNA constructs to directly observe fluctuations of the DNA backbone, while DNA constructs labeled with fluorescent DNA base analogues will be utilized to directly observe fluctuations of DNA bases. DNA breathing fluctuations should be greatly enhanced for bases in the vicinity of a replication fork junction, which should be confirmed at the single-molecule level by these measurements. A subsequent aim is to introduce the primosome helicase coded by bacteriophage T4 to these DNA replication fork constructs and observe how the DNA breathing fluctuations are affected by the presence and function of the helicase. It is hypothesized in this proposal that DNA breathing fluctuations are central to the helicase-catalyzed unwinding of dsDNA at a replication fork. If thi hypothesis is true, then one might expect to see significant changes in DNA breathing fluctuations when the helicase is present. The characterization of the interdependence of DNA breathing and helicase function will be a major step in the understanding of the mechanisms that drive DNA replication and transcription.