Consecutive ribonucleoside monophosphates on template inhibit DNA replication by T7 DNA polymerase or by T7 polymerase and helicase complex

rNTPs are structurally similar to dNTPs but are largely molar excessive in cell than dNTPs. rNTPs are inevitably incorporated into DNA to form rNMPs. Long RNA primers can also be incorporated into lagging-strand DNA. However, the influence of these incorporated rNMPs on T7 DNA replication remains unknown. In this work, we investigated primer extension past consecutive rNMPs (rA, r(AC), r(ACC), or r(ACCA)) on template by T7 DNA polymerase or by its complex with helicase. Primer extension is gradually inhibited with increasing rNMP number. rNMPs decrease the dNTP incorporation efficiency, slightly weaken the binding affinity of polymerase to DNA in ternary complex, and reduce the protein interaction between polymerase and helicase at DNA fork, thereby decreasing the fraction of the productive enzyme·DNA complex and the average primer extension rate. Therefore, the consecutive rNMPs on template gradually inhibit T7 primer extension and strand-displacement DNA synthesis, providing the kinetic information for the inhibition of rNMPs on DNA replication.