title: Effects of 2'-O-methyl nucleotide on ligation capability of T4 DNA ligase.
abstract: To further understand the ligation mechanism, effects of 2'-O-methyl nucleotide (2'-OMeN) on the T4 DNA ligation efficiency were investigated. Fluorescence resonance energy transfer assay was used to monitor the nick-joining process by T4 DNA ligase. Results showed that substitutions at 5'- and 3'-ends of the nick decreased the ligation efficiency by 48.7% ± 6.7% and 70.6% ± 4.0%, respectively. Substitutions at both 5'- and 3'-ends decreased the ligation efficiency by 76.6% ± 1.3%. Corresponding kinetic parameters, Vmax, Km, and kcat, have been determined in each case by using the Michaelis-Menten equation. The kinetic data showed that the 2'-OMeN substitutions reduced the maximal initial velocity and increased the Michaelis constant of T4 DNA ligase. Mismatches at 5'- and 3'-ends of the nick have also shown different influences on the ligation. Results here showed that the sugar pucker conformation at 3'-end impairs the ligation efficiency more profoundly than that at 5'-end. Different concentrations of Mg(2+), Ca(2+), K(+), Na(+), and ATP were also demonstrated to affect the T4 DNA ligase activity. These results enriched our knowledge about the effects of 2'-OMeN substitutions on the T4 DNA ligase.
authors.affiliation: Key Laboratory of Medical Cell Biology, Ministry of Education, Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001, China Department of Human Movement Science, Shenyang Sport University, Shenyang 110102, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Medical Cell Biology, Ministry of Education, Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001, China.
authors.forename: Zhaoxue
authors.initials: Z
authors.lastname: Tong
authors.affiliation: Key Laboratory of Medical Cell Biology, Ministry of Education, Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001, China.
authors.forename: Guojie
authors.initials: G
authors.lastname: Zhao
authors.affiliation: Department of Clinical Diagnosis, First Hospital of China Medical University, Shenyang 110001, China.
authors.forename: Runqing
authors.initials: R
authors.lastname: Mu
authors.affiliation: Department of Clinical Diagnosis, First Hospital of China Medical University, Shenyang 110001, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Shang
authors.affiliation: Key Laboratory of Medical Cell Biology, Ministry of Education, Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001, China yfguan55@sina.com.
authors.forename: Yifu
authors.initials: Y
authors.lastname: Guan
mesh_terms: D020137:Base Pair Mismatch; D011088:DNA Ligases / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D009711:Nucleotides / Q000378:metabolism*
chemical_list: D009711:Nucleotides; D011088:DNA Ligases
keywords: 2′-O-methyl nucleotide; T4 DNA ligase; ligation; mismatch