Location of the T4 Gene 32 Protein Binding Site on Simian Virus 40 DNA
Journal of Virology
The T4 gene 32 protein, which binds to single-stranded but not duplex DNA, forms a specifically located denaturation loop in covalently closed circular simian virus 40 (SV40) DNA. Cleavage of the SV40 DNA-gene 32 protein complex with a restriction endonuclease from Hemophilus parainfluenzae shows the loop center to be at 0.46 on the SV40 DNA map. This is within one of the regions of SV40 DNA cleaved preferentially by the single-strand-specific nuclease S,. Delius et al. (5) showed that
... howed that incubation of simian virus 40 (SV40) supercoiled DNA [SV40(I) DNA] with T4 gene 32 protein (1) under defined conditions followed by fixation with glutaraldehyde yields circular structures with one small "denaturation loop" per molecule. In an earlier paper (9), we reported that cleavage of such structures with the site-specific restriction endonuclease, EcoR, (6, R. N. Yoshimori, Ph.D. thesis, Univ. of California, San Francisco, 1971) yields linear molecules with the midpoint of the denaturation loop located 0.45 SV40 fractional length from one end. Because the two ends of the EcoR1-generated linear molecules are indistinguishable, it was impossible to assign a single fixed location for the T4 gene 32 protein binding site: that is, referring to the SV40 DNA map ( Fig. 1 ) (10), the denaturation loop could have occurred at 0.45 or 0.55 SV40 map units (fractional length from the EcoR, cleavage site), or both. To resolve this point, we have used a second site-specific restriction endonuclease, HpaII, to convert the gene 32 protein-SV40 DNA complexes to linear structures. HpaII cleaves SV40 DNA once at 0.73 on the SV40 map ( Fig. 1)(12) . Consequently, if the denaturation loop is located at 0.55 on the SV40 map, it will occur 0.18 SV40 fractional length from an end; alternatively, if the HpaII-generated linear molecules contain the denaturation loop 0.28 SV40 fractional length from an end, we could assign the T4 gene 32 protein binding site to 0.45 SV40 map position. Circular SV40 DNA with bound gene 32 protein was prepared (5) and dialyzed to remove glutaraldehyde for 3 h at 4 C against 10 mM Tris, pH 7.4, 1 mM EDTA, and 10 mM NaCl. After addition of MgCl2 to 10 mM and autoclaved gelatin to 100 jig/ml, an excess of HpaII restriction endonuclease was added and the mixture was incubated 1 h at 37 C. DNA was mounted for electron microscopy, and contour lengths were measured as previously described (9). The HpaII endonuclease was purified by a modification of the second method described by P. A. Sharp, B. Sugden, and J. Sambrook (Biochemistry, in press). Figure 2 shows the histogram of DNA lengths from the midpoint of the denaturation loop to the nearest end. Of 33 linear molecules containing T4 gene 32 protein that were scored by electron microscopy, 88% contained loops between 0. 25 and 0.29 SV40 fractional length from RI / a \M~~~~~~~A2+NDa Hp /M0.30.28 \~~~~~Ad2+ ND4 / 0.59 32 PROTEIN FIG. 1. Map of SV40 DNA. The RI restriction endonuclease cleavage site is the origin. Positions are expressed in SV40 DNA fractional length from the origin. The SV40 segment of Ad2+ND4 induces all known early, SV40 antigens and early SV40 RNA sequences (7, 8, 10). The regions of cleavage by single-strand-specific endonuclease S, are 0.45 to 0.55 (predominant at high ionic strength) and 0.15 to 0.25 (4).