Site specificity of psoralen-DNA interstrand cross-linking determined by nuclease Bal31 digestion.
A novel method for determination of psoralen photo-cross-linking sites in double-stranded DNA is described, which is based on a pronounced inhibition of Bal31 exonuclease activity by psoralen-DNA interstrand cross-links. The results using a 51 base pair fragment of plasmid pUC19 and a 346 base pair fragment of pBR322 show that 5'-TA sequences are preferred cross-linking sites compared to 3'-TA sequences. They also indicate that sequences flanking the 5'-TA site influence the cross-linking efficiency at the site. The DNA photo-cross-linking by 4,5',8-trimethylpsoralen and 8-methoxypsoralen was analyzed, and these two psoralens showed identical site specificity. The 5'-TA preference is rationalized on the basis of the local DNA structure in terms of the pi-pi electronic interaction between the thymines and the intercalated psoralens, as well as on the base tilt angles of the DNA.
D001483:Base Sequence; D004247:DNA; D004262:DNA Restriction Enzymes; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases / Q000378:metabolism; D011564:Furocoumarins; D010777:Photochemistry; D010957:Plasmids; D014307:Trioxsalen
D011564:Furocoumarins; D004247:DNA; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases; C038407:exonuclease Bal 31; D004262:DNA Restriction Enzymes; D014307:Trioxsalen