Photoreactive DNA Probes as a Tool for Studying the Translesion Synthesis System in Mammalian Cell Extracts

Abstract

Translesion synthesis (TLS) is one of the DNA damage tolerance strategies that has evolved to enable orga-nisms to replicate their genome despite the presence of unrepaired damage. TLS complexes are dynamic systems composed of DNA polymerases and associated protein factors. Therefore, it is hard to study these assembles by X-ray analysis or other instrumental methods. Here, we have suggested applying the photoaffinity labeling technique for studying the TLS system in nuclear/cellular extracts. As a tool we proposed to use partial DNA duplexes containing base-substituted photoreactive deoxynucleotides at the 3' end of primer opposite to DNA damage at the template strand. We demonstrated that photoreactive dNTPs can be potentially used to synthesize photoreactive DNA probes mimicking the DNA intermediates of the first stage of translesion synthesis by specialized DNA polymerases. We used synthetic apurinic/apyrimidinic site (AP-site) - tetrahydrofuran (THF) and 8 oxoguanine as damages in +1 position of the template strand with respect to 3' end of primer. Activity of human DNA polymerases beta and lambda was exploited for construction of photoreactive DNAs using photo derivatives of dNTPs. The kinetic parameters of the elongation reaction in model systems were estimated. Using photoaffinity crosslinking we found that only a few proteins in the bovine testis nuclear extract were strongly labeled by TLS probes.

Structural and Molecular Kinetic Features of Activities of DNA Polymerases

“…The photochemical properties of these groups are adjusted for the use of excitation near UV light, to avoid stimulation of the intrinsic photoreactivity of the nucleic acids and proteins. Upon photoirradiation, the generation of highly reactive species occurs, resulting in the direct covalent modification of adjacent molecules [ 34 , 35 , 36 , 37 ]. This approach has been successfully utilized to research the substrate-binding sites of RNA polymerase [ 38 ], of DNA polymerases [ 34 , 39 , 40 , 41 , 42 ], and of the complex of DNA polymerase α with primase [ 43 ].…”

“…Upon photoirradiation, the generation of highly reactive species occurs, resulting in the direct covalent modification of adjacent molecules [ 34 , 35 , 36 , 37 ]. This approach has been successfully utilized to research the substrate-binding sites of RNA polymerase [ 38 ], of DNA polymerases [ 34 , 39 , 40 , 41 , 42 ], and of the complex of DNA polymerase α with primase [ 43 ].…”