Binding of SSB and RecA protein to DNA-containing stem loop structures: SSB ensures the polarity of RecA polymerization on single-stranded DNA.
Single-stranded DNA-binding proteins play an important role in homologous pairing and strand exchange promoted by the class of RecA-like proteins. It is presumed that SSB facilitates binding of RecA on to ssDNA by melting secondary structure, but direct physical evidence for the disruption of secondary structure by either SSB or RecA is still lacking. Using a series of oligonucleotides with increasing amounts of secondary structure, we show that stem loop structures impede contiguous binding of RecA and affect the rate of ATP hydrolysis. The electrophoretic mobility shift of a ternary complex of SSB-DNA-RecA and a binary complex of SSB-DNA are similar; however, the mechanism remains obscure. Binding of RecA on to stem loop is rapid in the presence of SSB or ATPgammaS and renders the complex resistant to cleavage by HaeIII, to higher amounts of competitor DNA or low temperature. The elongation of RecA filament in a 5' to 3' direction is halted at the proximal end of the stem. Consequently, RecA nucleates at the loop and cooperative binding propagates the RecA filament down the stem causing its disruption. The pattern of modification of thymine residues in the loop region indicates that RecA monomer is the minimum binding unit. Together, these results suggest that SSB plays a novel role in ensuring the directionality of RecA polymerization across stem loop in ssDNA. These observations have fundamental implications on the role of SSB in multiple aspects of cellular DNA metabolism.
Reddy
M S
MS
Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.
Vaze
M B
MB
Madhusudan
K
K
Muniyappa
K
K
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D001667:Binding, Competitive; D004269:DNA, Bacterial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000037:antagonists & inhibitors / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006868:Hydrolysis; D008956:Models, Chemical; D009690:Nucleic Acid Conformation; D011108:Polymers / Q000378:metabolism; D011196:Potassium Permanganate / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011938:Rec A Recombinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013941:Thymine / Q000737:chemistry
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011108:Polymers; D011196:Potassium Permanganate; D011938:Rec A Recombinases; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases; D000251:Adenosine Triphosphatases; D013941:Thymine