Bam HI cleaves the self complementary dodecamer d-CGCGGAGCCGCG, before the two G's and possibly binds in the DNA major groove.
Oligodeoxynucleotides with GT or GA mispairs within the Bam HI recognition sequence (GGATCC), have been prepared. Binding and cleavage of the native vis a vis the mismatch substrates by Bam HI are analysed. UV melting curves and CD spectra of the oligomers suggest a double stranded B-DNA conformation. The enzyme Bam HI binds with varying affinities to the oligomers except the one with the GT wobble base pair. Bam HI cleaves the cognate sequence, GGATCC, between the two Guanines but cleaves GGAGCC before the guanines. The unusual cleavage is due to a local distortion in the DNA structure. Kinetic analysis of the cleavage reactions using the 35S labeled hexadecamers, d-ATGGCGGATCCGCCAT and d-ATGGCGGAGCCGCCAT, as substrates gives Km values 11.08 nM and 1.16 nM with corresponding Kcat of 11.04 and 0.62 min-1 respectively. The results are consistent with the binding of Bam HI in the major groove.
Roy
K B
KB
Centre for Biotechnology, Jawaharlal Nehru University, New Delhi, India.
Vrushank
D
D
D001482:Base Composition; D001483:Base Sequence; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D015245:Deoxyribonuclease BamHI / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D015183:Restriction Mapping; D013379:Substrate Specificity; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D004247:DNA; D015245:Deoxyribonuclease BamHI