Effect of cytarabine on the NMR structure of a model okazaki fragment from the SV40 genome.

Abstract

Okazaki fragments occur as intermediates during lagging strand DNA replication. Alterations in Okazaki fragment structure may contribute to the anticancer activities of nucleoside analogues such as cytarabine, a potent anti-leukemic agent that inhibits lagging strand replication. We have determined the solution structures for two model Okazaki fragments, [OKA] and [ARAC]. These sequences are derived from a frequent initiation site for primase during replication of the SV-40 viral genome. The sequence of [ARAC] differs from [OKA] only by substitution of cytarabine for one deoxycytidine. The structure of each model Okazaki fragment was elucidated using NMR spectroscopy and restrained molecular dynamics simulations. The solution structures of [OKA] and [ARAC] each consist of two distinct domains: a DNA duplex region (DDR) and an RNA-DNA hybrid duplex region (HDR). The DDR of [OKA] adopts geometry similar to B-form except for variations in helical parameters, especially twist and roll, which occur in the purine tract, increasing base overlap among the five consecutive purines. The helical axes for the DDR and HDR of [OKA] are bent 22 degrees relative to one another. Although the local structures for the DDR and HDR of [ARAC] are similar to those in [OKA] (root-mean-square deviation (rmsd) approximately 0.8, 1.7 A), the bending at the junction is different (41 degrees for [ARAC] vs 22 degrees for [OKA]). Increased helical bending of cytarabine-substituted Okazaki fragments may contribute to the propensity of cytarabine to inhibit elongation of the lagging strand during DNA replication, and in effecting anticancer activity.