Efficiency of T4 DNA ligase-catalyzed end joining after S1 endonuclease treatment on duplex DNA containing single-stranded portions.
Covalently closed-circular, superhelical SV4O DNA was used in all experiments. EcoRI endonuclease- and HpaII endonuclease-generated unit-length linear duplex DNAs were digested with S1 endonuclease under the conditions where single-stranded CNA was completely converted into the acid-soluble form. These were subjected to an end-to-end joining test with T4 DNA ligase. The ligation efficiency was significantly lower than that of the flush-ended linear duplex DNAs which were generated by both HpaI endonuclease digestion and the matching up of EcoRI-generated sticky end with Escherichia coli DNA polymerase I (Klenow fraction). However, the ligation efficiency of the S1-treated DNAs increased up to same level as the flush-ended DNA upon treatment with E. coli DNA polymerase I . Similar results were obtained in the case of S1 -generated unit-length linear duplex DNA. S1 does cleave both strands of superhelical DNA at unbasepaired sites.
D011088:DNA Ligases / Q000378:metabolism*; D004256:DNA Polymerase I / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D015246:Deoxyribonuclease EcoRI; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific; D004720:Endonucleases / Q000378:metabolism*; D011118:Polynucleotide Ligases / Q000378:metabolism*; D013539:Simian virus 40; D015719:Single-Strand Specific DNA and RNA Endonucleases
D004278:DNA, Superhelical; D004279:DNA, Viral; D004256:DNA Polymerase I; D004720:Endonucleases; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI; D019003:Deoxyribonuclease HpaII; C024406:endodeoxyribonuclease HpaI; D015252:Deoxyribonucleases, Type II Site-Specific; D015719:Single-Strand Specific DNA and RNA Endonucleases; D011088:DNA Ligases; D011118:Polynucleotide Ligases