ssDNA hybridization facilitated by T7 ssDNA binding protein (gp2.5) rapidly initiates from the strand terminus or internally followed by a slow zippering step

Abstract

The hybridization of two complementary ssDNA is essential for molecular biology and biological physics. T7 ssDNA binding protein (gp2.5) can rapidly facilitate this hybridization, but the mechanism and kinetic process remain unknown. As determined by fluorescence resonance energy transfer (FRET) and rapid kinetic analysis methods, gp2.5 binding coiling ssDNA to form gp2.5-ssDNA complex is the rate-limiting step for the entire DNA hybridization process. Afterward, the hybridization initiates from either the terminus or the internal part of ssDNA at a nucleation rate of 0.45 s^-1. The remaining DNA strands are hybridized in a zippering mode at a rate of 0.07 s^-1, limited by the dissociation of gp2.5 from ssDNA. The gp2.5-faciliated hybridization rate constant (74 μM^-1s^-1) is much higher than the spontaneous hybridization rate (1.7 μM^-1s^-1) in the absence of gp2.5. These hybridization mechanism and kinetic process are different from those by other ssDNA binding proteins, such as T4 gp32, Escherichia coli SSB protein and recA protein. Compare with E. coli SSB, the relatively slower association of gp2.5 to ssDNA and faster dissociation of gp2.5 from ssDNA are probably the major reasons to facilitate the rapid hybridization. This study provides valuable insights into the molecular mechanism of the recombination and repair processes and proposes a basis for improvement in their associated biotechnologies.

Keywords: FRET; Hybridization of complementary ssDNA; Rapid quench; T7 ssDNA binding protein gp2.5; Zipper hybridization.