The effects of substituted pyrimidines in DNAs on cleavage by sequence-specific endonucleases.

The rates of cleavage of DNAs containing substituents at position 5 of thymine or cytosine have been measured for a variety of sequence-specific endonucleases, so as to determine which features in the DNA sequence are being probed. Phage phi e DNA fully substituted with 5-hydroxymethyluracil is cleaved more slowly by enzymes whose recognition sequences contain A-T base pairs than are DNAs containing thymine, but both types of DNA are cleaved at similar rates by enzymes recognizing sequences composed only of G-C base pairs. Phage PBS2 DNA with uracil completely substituted for thymine is cleaved slowly by several enzymes which recognize sequences containing A-T base pairs (endonucleases Hpa I, HindII, and HindIII), while the rates of cleavage by other enzymes (endonucleases EcoRI and BamHI) are not affected. Phage lambda- and P22 DNAs containing 5-bromouracil are cleaved more slowly by several enzymes (endonucleases HindIII, Hpa I, BamHI) than are thymine-containing DNAs. Enzymes that recognize sequence isomers with the composition G:C:2A:2T (endonucleases EcoRI, Hpa I, HindIII) are not equally affected by substitution at position 5 of thymine, suggesting that they differ in their contacts with A-T base pairs. DNA containing glucosylated 5-hydroxymethylcytosine in place of cytosine is resistant to cleavage by all the endonucleases examined.