LASER-INDUCED PROTEIN-DNA CROSS-LINKS VIA PSORALEN FURANSIDE MONOADDUCTS

We have developed a technique for cross-linking DNA binding proteins t o 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 bi nding protein of Escherichia coli, T4 gp32, and RecA of E. coli) are s hown 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 o f cross-links. Titration experiments were carried out to measure the a pparent cross-linking constant (K(appXL)) for T7 RNA polymerase or Uvr B 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 invest igated as a function of the length of adducted DNA and also as a fract ion of the total noncovalent binding of proteins of psoralenated DNAs. The results showed that in the cases of T7 RNA polymerase and UvrB cr oss-linking was more efficient with short oligos (8 and 19 mers) as co mpared to longer oligos (50 mer). A tryptic peptide of T7 RNA polymera se that was conjugated to a psoralen furanside monoadducted 12 mer DNA was isolated by high-performance liquid chromatography. Mass spectrom etry and amino acid composition of this peptide revealed that it origi nated from a region between residues 558 and 608 of the primary struct ure of T7 RNA polymerase. Two other peptides cross-linked to oligos we re 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 t he proteins. We have shown that T7 RNA polymerase molecules in a terna ry complex arrested at the furanside monoadduct can be cross-linked to the DNA templates with laser light. Evidence suggests that the arrest ed polymerase molecules existed in multiple conformations on the DNA t emplate. This method of transcriptional cross-linking offers a new met hod for preparing highly stable elongation complexes for further studi es.