A specific partner for abasic damage in DNA

In most-models of DNA replication, Watson-Crick hydrogen bonding drives the incorporation of nucleotides into the new strand of DNA and maintains the complementarity of bases with the template strand. Studies with nonpolar an alogues of thymine and adenine, however, have shown that replication is sti ll efficient in the absence of hydrogen bonds(1-4). The replication of base pairs might also be influenced by steric exclusion, whereby inserted nucle otides need to be the correct size and shape to fit the active site against a template base(5,6). A simple steric-exclusion model map not require Wats on-Crick hydrogen bonding to explain the fidelity of replication, nor shoul d canonical purine and pyrimidine shapes be necessary for enzymatic synthes is of a base pair if each can fit into the DNA double helix without steric strain(6). Here we test this idea by using a pyrene nucleoside triphosphate (dPTP) in which the fluorescent 'base' is nearly as large as an entire Wat son-Crick base pair. We show that the non-hydrogen-bonding dPTP is efficien tly and specifically inserted by DNA polymerases opposite sites that lack D NA bases. The efficiency of this process approaches that of a natural base pair and the specificity is 10(2)-10(4)-fold. We use these properties to se quence abasic lesions in DNA, which are a common form of DNA damage in vivo (7). In addition to their application in identifying such genetic lesions, our results show that neither hydrogen bonds nor purine and pyrimidine stru ctures are required to form a base pair with high efficiency and selectivit y. These findings confirm that steric complementarity is an important facto r in the fidelity of DNA synthesis.