Laser-induced protein-DNA cross-links via psoralen furanside monoadducts.
We have developed a technique for cross-linking DNA binding proteins to DNA using psoralen furanside monoadducts as photoaffinity probes and a continuous-wave argon ion laser (366 nm) as a light source. Several DNA binding proteins (T7 RNA polymerase, UvrB, single-stranded DNA binding protein of Escherichia coli, T4 gp32, and RecA of E. coli) are shown to cross-link to single-stranded psoralen monoadducted DNA oligos differing in length and sequence. Increasing fluences of laser light on a fixed ratio of DNA/protein resulted in an increase in the yield of cross-links. Titration experiments were carried out to measure the apparent cross-linking constant (KappXL) for T7 RNA polymerase or UvrB to a monoadducted 24 mer DNA. The estimated values for the apparent cross-linking constant were in the range of (2-3) x 10(-7) M for both T7 RNA polymerase and UvrB. The efficiency of cross-linking was investigated as a function of the length of adducted DNA and also as a fraction of the total noncovalent binding of proteins of psoralenated DNAs. The results showed that in the cases of T7 RNA polymerase and UvrB cross-linking was more efficient with short oligos (8 and 19 mers) as compared to longer oligos (50 mer). A tryptic peptide of T7 RNA polymerase that was conjugated to a psoralen furanside monoadducted 12 mer DNA was isolated by high-performance liquid chromatography. Mass spectrometry and amino acid composition of this peptide revealed that it originated from a region between residues 558 and 608 of the primary structure of T7 RNA polymerase. Two other peptides cross-linked to oligos were also purified. Repeated attempts to perform Edman sequencing of the peptide-DNA conjugates failed. Overall evidence indicates that photo-cross-linking of furanside monoadducts occurred at multiple sites on the proteins. We have shown that T7 RNA polymerase molecules in a ternary complex arrested at the furanside monoadduct can be cross-linked to the DNA templates with laser light. Evidence suggests that the arrested polymerase molecules existed in multiple conformations on the DNA template. This method of transcriptional cross-linking offers a new method for preparing highly stable elongation complexes for further studies.
Sastry
S S
SS
Department of Chemistry, University of California, Berkeley.
Spielmann
H P
HP
Hoang
Q S
QS
Phillips
A M
AM
Sancar
A
A
Hearst
J E
JE
D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D007834:Lasers; D008027:Light; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D014307:Trioxsalen / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D014357:Trypsin
D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; C016507:hydroxymethyltrioxsalen; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D014357:Trypsin; D014307:Trioxsalen