Replication by a single DNA polymerase of a stretched single-stranded DNA

A new approach to the study of DNA/protein interactions has been opened thr ough the recent advances in the manipulation of single DNA molecules. These allow the behavior of individual molecular motors to be studied under load and compared with bulk measurements. One example of such a motor is the DN A polymerase, which replicates DNA. We measured the replication rate by a s ingle enzyme of a stretched single strand of DNA. The marked difference bet ween the elasticity of single- and double-stranded DNA allows for the monit oring of replication in real time. We have found that the rate of-replicati on depends strongly on the stretching force applied to the template. In par ticular, by varying the load we determined that the biochemical steps limit ing replication are coupled to movement. The replication rate increases at low forces, decreases at forces greater than 4 pN, and ceases when the sing le-stranded DNA substrate is under a load greater than approximate to 20 pN . The decay of the replication rate follows an Arrhenius law and indicates that multiple bases on the template strand are involved in the rate-limitin g step of each cycle. This observation is consistent with the induced-fit m echanism for error detection during replication.