Photoaffinity Labeling Technique for Studying DNA Replication and DNA Repair†

Abstract

The photoaffinity labeling technique is based on UV-light induced crosslinking of proteins to photoreactive DNA. Photoreactive groups can be introduced at different points of DNA (base or phosphate) either at the ends or in inner positions of the DNA chain by combination of enzymatic and chemical synthesis. Varying the structure of photoreactive DNA one can design the intermediates of different stages of DNA replication or DNA repair and apply them to identify the proteins crosslinked to specific positions of the DNA chain in single- or double-stranded DNA, partial DNA duplexes, gapped or nicked duplexes or DNA carrying flap structures. A wide range of base-substituted dNTP analogs containing photoreactive groups of different photoreactivity and spacers of various lengths has been synthesized and characterized. Photoreactive dNTP analogs have been shown to be effective substrates of viral, bacterial and eukaryotic DNA polymerases. The efficiencies of crosslinking of DNA bearing various photoreactive dNMP to protein and DNA targets were estimated. New approaches for enzymatic introduction of photoreactive groups onto the 5'-end of oligonucleotides and into the inner positions of DNA chain have been elaborated. Photoreactive DNAs have been successfully used to study enzymes and protein factors of DNA replication and repair in reconstituted systems and cellular/nuclear extracts. Photoaffinity labeling technique was shown to be a prominent tool of proteomics to elucidate structural and functional aspects of protein-DNA interactions. It can be also applied for identification of proteins including unknown ones, which interact with specific DNA intermediates in cellular/nuclear extracts.