Molecular Weight Determination of DNA-Binding Proteins by UV Cross-linking Combined with SDS Polyacrylamide Gel Electrophoresis

It was first shown by Smith (1962) that UV-light irradiation of E. coli decreased the amount of DNA that could be extracted by sodium dodecylsulphate in the presence of 0.5 M KCl. The remaining DNA was cross-linked with protein, forming insoluble complexes. Later Smith et al. ( 1966, 1968, 1969, 1970) showed that UV-light caused formation of a covalent linkage between cysteine and uracil (5-s-cysteine-6-hydrouracil) , poly U, poly C and thymine. Among L-amino acids, the most reactive with uracil are phenylalanine, tyrosine and cysteine (Smith, 1969). The photochemical attachment of specific proteins to DNA could be later improved by using bromodeoxyuridine-substituted DNA (Lin and Riggs, 1979). UV irradiation of BrdU-substituted DNA leads to debromination and the subsequent production of highly reactive uracilyl radicals (Smith and Hanawalt, 1969). Proteins, therefore, will be cross-linked more readily to BrdU-DNA than to unsubstituted DNA. The formation of such stable complexes between the radioactively labelled DNA and the protein enables estimation of the molecular weight of the protein in relatively crude preparations. This method has been used with success to determine the molecular weight of specific proteins binding to double-stranded DNA, singlestranded DNA and RNA (Hillel and Wu, 1978, Chodosh et al., 1986, Jiricny et al., 1988, Feavers et al., 1989).