Efficient synthesis of a supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication.
We report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, we report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. We demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.
Kodadek
T
T
Department of Chemistry, University of Texas, Austin 78712.
Gamper
H
H
D001483:Base Sequence; D003090:Coliphages / Q000235:genetics; D004247:DNA / Q000138:chemical synthesis*; D018736:DNA Adducts; D004261:DNA Replication; D004278:DNA, Superhelical / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D011564:Furocoumarins / Q000138:chemical synthesis*; D013604:T-Phages / Q000235:genetics; D014307:Trioxsalen / Q000031:analogs & derivatives; D014764:Viral Proteins / Q000378:metabolism
D018736:DNA Adducts; D004278:DNA, Superhelical; D011564:Furocoumarins; D014764:Viral Proteins; C056465:psoralen-DNA adduct; C016507:hydroxymethyltrioxsalen; D004247:DNA; D014307:Trioxsalen