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 degrees, in reasonably good agreement with the bend angle determined by transient electric birefringence, 38 degrees +/- 7 degrees. The normal 476-bp DNA fragment exhibited a Gaussian distribution of bend angles centered at 0 degrees, 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.

FIGURE 1

The noncoding intergenic M13 origin of replication, which is found in the plasmid LITMUS 28 from 1043 to 1545 bp (between the HpaI and HincII cut sites). Highlighted are the ribosome binding site at 1043–1056 bp, the Pribnow box (−10) and −35 hexamer, shown as boxes ∼8 bp and 35 bp from the gpII mRNA start site at 1077 bp, the start site for replication of complementary (minus) strand DNA synthesis ((−) ori RNA) at 1265 bp and the viral (plus) strand initiation site ((+) ori DNA) at 1291 bp, which are indicated by arrows, and the gpII nick site at 1266 bp. The sequence between 925 and 1042 bp belongs to the ampicillin resistance gene. The 471-bp fragment used in this study was obtained by using the restriction enzymes AhdI/BanII to digest the plasmid at 931 and 1402 bp.

FIGURE 2

(a) The logarithm of DNA molecular weight, N, in basepairs, as a function of the absolute electrophoretic mobility, μ, observed for the 471-bp fragment (□) and the 476-bp fragment (Δ) in a 6.9% T, 3% C polyacrylamide gel cast and run in 40 mM Tris/1 mM EDTA/39.2 mM MgCl₂, pH 8.0 at 23°C using E = 3.33 V/cm. The solid line and circles correspond to the mobilities observed for a standard 50-bp ladder run in the same gel. (b) Semilogarithmic plot of the normalized decay of the birefringence as a function of time. The circles correspond to the measured data for the 471-bp fragment in the buffer (1 mM Tris/0.1 mM EDTA, pH 8.0) at 20°C. The dashed line corresponds to a two-exponential fit of the data points. Only the fitted curve is shown for the 476-bp fragment (solid line) and a normal, unbent 471-bp fragment (dashed-dot line).

FIGURE 3

AFM images of DNA molecules in air in mica. Left, the 476-bp fragment; right, the 471-bp fragment. AFM was operated in tapping mode with a scan scale of 1 × 1 μm.

FIGURE 4

Histograms of the frequency of occurrence of end-to-end distances: (a) for the 476-bp fragment and (b) for the 471-bp fragment. The molecules measured for the analysis were 73 for the 476-bp fragment and 69 for the 471-bp fragment, respectively. The measured mean-square end-to-end distance, 〈R²〉, was converted into the percentage of the average measured contour length of the target DNA.

FIGURE 5

Histograms of the frequency of occurrence of bend angles: (a) for the 476-bp fragment and (b) for the 471-bp fragment. The same numbers of molecules were analyzed as given in Fig. 3.