Formation of nucleoprotein RecA filament on single-stranded DNA. Analysis by stepwise increase in ligand complexity.
RecA protein plays a pivotal role in homologous recombination in Escherichia coli. RecA polymerizes on single-stranded (ss) DNA forming a nucleoprotein filament. Then double-stranded (ds) DNA is bound and searched for segments homologous to the ssDNA. Finally, homologous strands are exchanged, a new DNA duplex is formed, and ssDNA is displaced. We report a quantitative analysis of RecA interactions with ss d(pN)n of various structures and lengths using these oligonucleotides as inhibitors of RecA filamentation on d(pT)20. DNA recognition appears to be mediated by weak interactions between its structural elements and RecA monomers within a filament. Orthophosphate and dNMP are minimal inhibitors of RecA filamentation (I50 = 12-20 mM). An increase in homo-d(pN)2-40 length by one unit improves their affinity for RecA (f factor) approximately twofold through electrostatic contacts of RecA with internucleoside phosphate DNA moieties (f approximately = 1.56) and specific interactions with T or C bases (f approximately = 1.32); interactions with adenine bases are negligible. RecA affinity for d(pN)n containing normal or modified nucleobases depends on the nature of the base, features of the DNA structure. The affinity considerably increases if exocyclic hydrogen bond acceptor moieties are present in the bases. We analyze possible reasons underlying RecA preferences for DNA sequence and length and propose a model for recognition of ssDNA by RecA.
Bugreeva
Irina P
IP
Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia.
Bugreev
Dmitry V
DV
Nevinsky
Georgy A
GA
D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D008024:Ligands; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011938:Rec A Recombinases / Q000378:metabolism*; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D008024:Ligands; D011938:Rec A Recombinases