O N Voloshin1, R D Camerini-Otero. 1. Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health, Bethesda, MD 20892-1810, USA.
Abstract
BACKGROUND: The RecA protein is a central player in bacterial homologous recombination. It promotes two key events: the search for homology between two DNA molecules and the subsequent formation of the synaptic complex composed of RecA and three DNA strands (two from one duplex and one single strand). In spite of numerous studies, the architecture of the synaptic complex is still far from clear. RESULTS: We have exploited two approaches to study the structure of Escherichia coli RecA protein-mediated DNA synapsis: chemical modification with potassium permanganate and treatment of synaptic complexes of different lengths with topoisomerase I. The linking number difference values, obtained after separation of the individual sets of topoisomers in an agarose gel, were used to determine the number of bases per helical turn. We were able to show that the topology of the three-stranded complexes containing RecA is quite different from that expected for deproteinized D-loops. The original duplex in the synaptic complex is unwound, but not necessarily unpaired, to a structure topologically equivalent to DNA with approximately 27 bp per turn. Despite the fact that the patterns of reactivity towards potassium permanganate cannot be interpreted unambiguously, the results of chemical footprinting can be explained in terms of a synaptic complex as an extended and unwound three-stranded helical structure. CONCLUSIONS: This work provides the first quantitative topological parameters for the RecA protein-mediated three-stranded synaptic complex. Furthermore, the structure of synaptic complexes is different from that of a simple D-loop.
BACKGROUND: The RecA protein is a central player in bacterial homologous recombination. It promotes two key events: the search for homology between two DNA molecules and the subsequent formation of the synaptic complex composed of RecA and three DNA strands (two from one duplex and one single strand). In spite of numerous studies, the architecture of the synaptic complex is still far from clear. RESULTS: We have exploited two approaches to study the structure of Escherichia coli RecA protein-mediated DNA synapsis: chemical modification with potassium permanganate and treatment of synaptic complexes of different lengths with topoisomerase I. The linking number difference values, obtained after separation of the individual sets of topoisomers in an agarose gel, were used to determine the number of bases per helical turn. We were able to show that the topology of the three-stranded complexes containing RecA is quite different from that expected for deproteinized D-loops. The original duplex in the synaptic complex is unwound, but not necessarily unpaired, to a structure topologically equivalent to DNA with approximately 27 bp per turn. Despite the fact that the patterns of reactivity towards potassium permanganate cannot be interpreted unambiguously, the results of chemical footprinting can be explained in terms of a synaptic complex as an extended and unwound three-stranded helical structure. CONCLUSIONS: This work provides the first quantitative topological parameters for the RecA protein-mediated three-stranded synaptic complex. Furthermore, the structure of synaptic complexes is different from that of a simple D-loop.
Authors: Alexander A Volodin; Tatiana N Bocharova; Elena A Smirnova; R Daniel Camerini-Otero Journal: J Biol Chem Date: 2008-11-11 Impact factor: 5.157
Authors: Dmitry V Bugreev; Roberto J Pezza; Olga M Mazina; Oleg N Voloshin; R Daniel Camerini-Otero; Alexander V Mazin Journal: Nat Struct Mol Biol Date: 2010-12-12 Impact factor: 15.369