Study of MMLV RT- binding with DNA using surface plasmon resonance biosensor.
Surface plasmon resonance biosensor technique was used to study the binding of Moloney murine leukemia virus reverse transcriptase without RNase H domain (MMLV RT-) with DNA in the absence and in the presence of inhibitors. Different DNA substrates, including single-stranded DNA (ssDNA), DNA template-primer (T-P) duplex and gapped DNA, were immobilized on the biosensor chip surface using streptavidin-biotin, and MMLV RT(-)-DNA binding kinetics were analyzed by different models. MMLV RT-; could bind with ssDNA and the binding was involved in conformation change. MMLV RT-; binding DNA T-P duplex and gapped DNA could be analyzed using the simple 1:1 Langmuir model. The lack of RNase H domain reduced the affinity between MMLV RT-; and T-P duplex. The effects of RT inhibitors, including efavirenz, nevirapine and quercetin, on the interaction between MMLV RT-; and gapped DNA were analyzed according to recovered kinetics parameters. Efavirenz slightly interfered with the binding between RT and DNA and the affinity constant in the presence of the inhibitor (K(A) = 1.21 x 10(6) M(-1)) was lower than in the absence of the inhibitor (KA = 4.61 x 10(6) M(-1)). Nevirapine induced relatively tight binding between RT and DNA and the affinity constant in the presence of the inhibitor (K(A) = 1.47 x 10(7) M(-1)) was approximately three folds higher than without nevirapine, mainly due to rapid association and slow dissociation. Quercetin, a flavonoid originating from plant which has previously shown strong inhibition of the activity of RT, was found to have minimal effect on the RT-DNA binding.
Wu
Lei
L
Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China.
Huang
Ming-Hui
MH
Zhao
Jian-Long
JL
Yang
Meng-Su
MS
D000480:Alkynes; D048588:Benzoxazines; D003521:Cyclopropanes; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D004277:DNA, Single-Stranded / Q000378:metabolism; D007700:Kinetics; D008979:Moloney murine leukemia virus / Q000201:enzymology*; D019829:Nevirapine / Q000494:pharmacology; D010078:Oxazines / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000502:physiology; D011794:Quercetin / Q000494:pharmacology; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology; D016914:Ribonuclease H / Q000235:genetics; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance; D013698:Templates, Genetic
D000480:Alkynes; D048588:Benzoxazines; D003521:Cyclopropanes; D017931:DNA Primers; D004277:DNA, Single-Stranded; D010078:Oxazines; D018894:Reverse Transcriptase Inhibitors; D004247:DNA; D019829:Nevirapine; D011794:Quercetin; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; C098320:efavirenz