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The reaction mechanism of the DNA repair enzyme bacteriophage T4 endonuclease V involves a rate limiting beta-elimination step chemical reaction catalyzed by an unknown amino acid residue of T4 not by an internucleotide phosphate. Substrates of modified oligodeoxyribonucleotide duplexes having a cis-syn thymine dimer were used to analyze the reaction, showing that a stable complex is formed by the completely changed enzyme molecules.

The interactions between T4 endonuclease and its substrate is observed by using synthetic DNA duplexes having a cis-syn thymine dimer. Oligonucleotides having a methylphosphonate linkage at the thymine dimer site are prepared and their binding with the enzyme is analyzed by the filter-binding method. Changes in the minor groove of DNA duplexes result in the reduction of enzyme binding. The substrate analogue with a methylphosphonate linkage is cleaved for R(sub P) configuration and not for S(sub P) configuration.