Structural Stability of Rad51 Filaments of Self-Aggregates and of Presynaptic Complexes Studied by Electron Microscopy and Pressure Tuning Fluorescence Spectroscopy

Abstract

The maintenance and stability of the genetic material of cells is of vital significance. This task is fulfilled by complex mechanisms involving the associates of specific proteins that through several steps recognize and repair DNA damages. In this study we focus our attention on an early step in the process of homologous recombination of human cells that is related to the function of the protein Rad51. The repair action of the protein is initiated by a supposedly unspecific helical filament formation of its monomers around single stranded DNA overhangs at double strand breaks. We study the conditions that regulate the structure and thus the biological activity of this, presynaptic” filament. Now we report results of pressure tuning fluorescence spectroscopy (Schay et al.(2006) JBC, 281, 25972) concerning the strength of interaction at the monomer-monomer interfaces in the filament structures and results of electronmicroscopy concerning their topology and structural parameters. The fluorescence signal is based on the dye ANS that we show being bound near the monomer interfaces. The measurements yield new data concerning the role of ATP, Mg, and Ca as cofactors of the presynaptic filament formation and show the unique effect of bound K-ions significantly influencing both the stability and the topology of the presynaptic as well as of the self-aggregate filaments.Acknowledgements: the authors are grateful to Prof. Kovacs of Eotvos University for help and valuable advices in electron microscopy. The work was supported by the Hungarian grant OTKA K 84271.