Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Structure of DNA Four-Way Junctions: Effect of Ions and Proteins
Holliday or DNA four-way junctions are important intermediates in recombination and repair processes (1-4). These structures can change conformation rapidly in solution interconverting from an open, four-fold symmetrical structure, with no central base stacking to one in which coaxial stacking of the helical arms has been observed (5). The open structure, which is capable of branch migration, is functionally relevant; however, many proteins have been observed to bind to and stabilize the stacked form. Our investigations have focused on elucidating the conformational changes induced by the binding of ions and the architectural proteins, HU and IHF, to improve understanding of the stacked form of the junction. Using fluorescence spectroscopic methods, we have examined the ion-binding site and have explicitly explored the coordination of ions to the central region of the junction. Förster resonance energy transfer (FRET) experiments have revealed that the degree of stacking or interduplex angle (IDA) of the junction is modulated by ion size, where larger ionic radii lead to larger IDAs. The proteins, HU and IHF, which are known to bind and bend DNA, stabilize the junction in the stacked conformation and induce a greater degree of stacking upon binding (6). In contrast, the repair protein, Msh2-Msh6, induces the junction to adopt an open conformation. All three proteins recognize and bind to the junction structure with nanomolar affinity and interestingly, Msh2-Msh6 binds the junction with higher affinity than a mismatch site. A novel FRET-mapping approach has been employed to determine the location of these proteins on the junction and indicates that the proteins bind to the central region of the junction.
This research has been supported by grants (MCB-0316625; MCB-0843656) from the NSF awarded to I.M.
C. Iulia Vitoc
Molecular Biology and Biochemistry Department