Incorporation of Dihydrothymidine and its Triphosphate During DNA Replication: An Implication for the Biological Consequence of Thymine C5-C6 Bond Saturation
It has been shown by extensive product analysis that the saturation of the C5-C6 bond of thymine and the concomitant loss of planarity of the thymine ring are notable characteristics of the DNA lesions produced by ionizing radiation (1). Recently, we (2) and other laboratories (3–5) have found that thymine glycols present in a DNA template constitute replicative blocks to DNA synthesis in vitro. Interestingly a base, presumably adenine, is incorporated opposite thymine glycol; however, DNA synthesis is arrested at the site. In order to obtain a more complete picture of the biological consequence of thymine C5-C6 bond saturation, we have focused on dihydrothymine. Although dihydrothymine is produced by ionizing radiation preferentially under anoxic conditions (6.7), it shares common structural characteristics with thymine glycol: the thymine rings of both compounds are no longer planar due to the saturation of C5-C6 bond and assume a half chair conformation with C5 and C6 significantly out of the plane of the other four ring atoms (8). Since it is difficult to selectively introduce dihydrothymine into DNA by ionizing radiation or chemical reagents, we have used the following alternative approaches. In the first approach, DNA containing dihydrothymine was prepared by in vitro DNA synthesis catalyzed by DNA polymerase in the presence of dihydrothymidine 5’-triphosphate (DHdTTP). In the second approach, we developed an in vivo system in which exogeneously added dihydro thymidine was incorporated into DNA. Thus we could obtain information not only about the coding property of dihydrothymine and the effect of dihydrothymine on the local structure of DNA, but also about the initial interactions of the molecule with DNA polymerases.
KeywordsMicrococcus Luteus Common Structural Characteristic Half Chair Conformation Replicative Block Prime Terminus
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