Enzymatic analysis of isomeric trithymidylates containing ultraviolet light-induced cyclobutane pyrimidine dimers. I. Nuclease P1-mediated hydrolysis of the intradimer phosphodiester linkage.
Our recent findings suggest that enzymatic hydrolysis of the intradimer phosphodiester bond may constitute the initial step in the repair of UV light-induced cyclobutane pyrimidine dimers in human cells. To examine the susceptibility of this phosphodiester linkage to enzyme-mediated hydrolysis, the trinucleotide d-Tp-TpT was UV-irradiated and the two isomeric compounds containing a cis-syn-cyclobutane dimer were isolated by high performance liquid chromatography and treated with various deoxyribonucleases. Snake venom phosphodiesterase hydrolyzed only the 3'-phosphodiester group in the 5'-isomer (d-T less than p greater than TpT) but was totally inactive toward the 3'-isomer (d-TpT less than p greater than T). In contrast, calf spleen phosphodiesterase only operated on the 3'-isomer by cleaving the 5'-internucleotide bond. Kinetic analysis revealed that (i) the activity of snake venom phosphodiesterase was unaffected by a dimer 5' to a phosphodiester linkage, (ii) the action of calf spleen phosphodiesterase was partially inhibited by a dimer 3' to a phosphodiester bond, and (iii) Escherichia coli phr B-encoded DNA photolyase reacted twice as fast with d-T less than p greater than TpT as with d-TpT less than p greater than T. Mung bean nuclease, nuclease S1, and nuclease P1 all cleaved the 5'-internucleotide linkage, but not the intradimer phosphodiester bond, in d-TpT less than p greater than T. Both phosphate groups in d-T less than p greater than TpT were refractory to mung bean nuclease or nuclease S1. Incubation of d-T less than p greater than TpT with nuclease P1, however, generated the novel compound dT less than greater than d-pTpT containing a severed intradimer phosphodiester linkage. Accordingly, nuclease P1 represents the first purified enzyme known to hydrolyze an intradimer phosphodiester linkage.
Liuzzi
M
M
Department of Medicine, Cross Cancer Institute, Edmonton, Alberta, Canada.
Weinfeld
M
M
Paterson
M C
MC
D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D004277:DNA, Single-Stranded / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D066298:In Vitro Techniques; D007536:Isomerism; D009708:Nucleotidases / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010777:Photochemistry; D011740:Pyrimidine Dimers / Q000032:analysis / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013938:Thymidine Monophosphate / Q000528:radiation effects*; D013942:Thymine Nucleotides / Q000528:radiation effects*; D014466:Ultraviolet Rays
D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D011740:Pyrimidine Dimers; D013942:Thymine Nucleotides; D013938:Thymidine Monophosphate; D003851:Deoxyribonucleases; D009708:Nucleotidases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D010727:Phosphoric Diester Hydrolases