Analysis of the Intrinsic Bend in the M13 Origin of Replication by Atomic Force Microscopy

Abstract

Atomic force microscopy (AFM) has been used to image a 471-bp bent DNA restriction fragment derived from the M13 origin of replication in plasmid LITMUS 28, and a 476-bp normal, unbent fragment from plasmid pUC19. The most probable angle of curvature of the 471-bp DNA fragment is 40–50°, in reasonably good agreement with the bend angle determined by transient electric birefringence, 38° ± 7°. The normal 476-bp DNA fragment exhibited a Gaussian distribution of bend angles centered at 0°, indicating that this fragment does not contain an intrinsic bend. The persistence length, P, was estimated to be 60 ± 8 and 62 ± 8 nm for the 471- and 476-bp fragments, respectively, from the observed mean-square end-to-end distances in the AFM images. Since the P-values of the normal and bent fragments are close to each other, the overall flexibility of DNA fragments of this size is only marginally affected by the presence of a stable bend. The close agreement of AFM and transient electric birefringence results validates the suitability of both methods for characterizing DNA bending and flexibility.