Research Of Transforming Linear DNA To Covalently Closed Circular DNA By T4 DNA Ligase

T4 DNA Ligase(T4Lig)can catalyze adjacent 3'OH and 5'PO4of DNA to form 3'-5'-phosphodiester bond.T4 Lig is widely used in cloning,sequencing and gene synthesis.Presently,the ligation mechanism of T4 Lig is using nicked DNA(n DNA)as substrate.But the ligation mechanism of linear DNA(L-DNA)as substrate by T4 Lig is pooly understood.In the conventional ligation,it is widely believed that L-DNA can be transformed to covalently closed circular DNA(ccc DNA).This thought is incompatible with the phenomenon that the transformants of L-DNA ligation product always lack nucleotides in ligation site.This research built the p ET22b-S4 plasmid containing xylanase gene S4.We amplify the plasmid to get the blunt-end L-DNA by PCR,and digest the plasmid to get the sticky-end L-DNA by Xho I.The blunt-end or sticky-end L-DNA are ligated three times by T4 Lig to get ccc DNA.The results of research are as follows:1.We prove that the conventional ligation prouduct of L-DNA is n DNA but not ccc DNA: 1)When the blunt-end L-DNA is ligated intra molecular by T4 Lig,part transformants always lack nucleotides in ligation site.But the double strands of ccc DNA are intact that will not lack nucleotides,so the ligation prouducts of L-DNA is not ccc DNA.2)The electrophoresis bands of ligation prouducts between dilateral and unilateral phosphorylated L-DNA is uniform.The ligation prouducts of unilateral phosphorylated blunt-end L-DNA go through agarose gel electrophoresis,each band is recycled,and sequenced by Sanger DNA chain termination method to determine the bands are L-DNA,n DNA and dimer DNA,but no ccc DNA.This proves that the ligation prouduct of blunt-end L-DNA is n DNA.3).The electrophoresis bands of ligation prouducts by sticky-end L-DNA are same with the blunt-end L-DNA.This proves that the ligation prouduct of sticky-end L-DNA is n DNA.4).The ligation prouduct reacts with the T5 exonuclease,and all the bands are degraded,further proving that the ligation prouduct of L-DNA is n DNA.2.The AMP in the nick prevents further ligation of n DNA : 1)The ligation prouduct of L-DNA is heated or purifid to remove T4 Lig.And then begin the second conventional by T4 Lig,but there is still no ccc DNA.It's proved that not T4 Lig prevents the ligation.Maybe the 5'-PO4 in the nick is adenylated,which means 5'-PO4 is combined with the AMP to prevent further ligation.2).Try to use RNase H?toreact with the ligation product to remove AMP,then begin second conventional ligation again,but there is no change of n DNA,which proves that 5'-PO4 is combined with AMP by pyrophosphate bond,rather than phosphodiester bond.3.Transform n DNA to ccc DNA: 1)The n DNA of conventional ligation product is ligated by second ligation without ATP using T4 Lig,and produces a small amount of ccc DNA.It proves that deadenylated T4 Lig can ligate the n DNA.2)The second ligation by T4 Lig without ATP removes AMP,then a third conventional ligation is conducted,which appears more ccc DNA.3)The 159 lysine is concerned with adenosine,so mutant K159 L can't be adenylatd by ATP,but can combine the nick containing AMP.Remove the AMP by K159 L in second ligation,then a third conventional ligation is conductd,which transforms 90% n DNA to ccc DNA.We prove that the conventional ligation prouduct of L-DNA is n DNA.The AMP prevents n DNA further form ccc DNA.Using K159 L or T4 Lig without ATP in second ligation to remove AMP,then transform n DNA to ccc DNA by the third conventional ligation.This study further understands the ligation mechanism of L-DNA by T4 Lig.And corrects the mistaken understanding of ligation in L-DNA.The results of this research are great significant for the application of T4 Lig.