B-DNA to Z-DNA structural transitions in the SV40 enhancer: Stabilization of Z-DNA in negatively supercoiled DNA minicircles

• Published in: Biochemistry (Easton) Vol. 32; no. 9; pp. 2167 - 2176
• Main Authors: Gruskin, Elliott A, Rich, Alexander
• Format: Journal Article
• Language: English
• Published: Legacy CDMS American Chemical Society 09.03.1993
• Subjects: Biological and medical sciences; Conformational dynamics in molecular biology; DNA, Superhelical - chemistry; DNA, Viral - chemistry; Enhancer Elements, Genetic; Exobiology; Fundamental and applied biological sciences. Psychology; Molecular biophysics; Nucleic Acid Conformation; Simian virus 40 - genetics; Space life sciences; Non-Nasa Center; Nasa Discipline Exobiology; Nasa Discipline Number 52-20; Nasa Program Exobiology; Virus; Z DNA; Enhancer sequence; Superhelical structure; Conformational dynamics; Polyomavirus; Papovaviridae; SV40 virus; Conformational transition; NASA Discipline Exobiology; NASA Program Exobiology; NASA Discipline Number 52-20; Non-NASA Center
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00060a007

During replication and transcription, the SV40 control region is subjected to significant levels of DNA unwinding. There are three, alternating purine-pyrimidine tracts within this region that can adopt the Z-DNA conformation in response to negative superhelix density: a single copy of ACACACAT and two copies of ATGCATGC. Since the control region is essential for both efficient transcription and replication, B-DNA to Z-DNA transitions in these vital sequence tracts may have significant biological consequences. We have synthesized DNA minicircles to detect B-DNA to Z-DNA transitions in the SV40 enhancer, and to determine the negative superhelix density required to stabilize the Z-DNA. A variety of DNA sequences, including the entire SV40 enhancer and the two segments of the enhancer with alternating purine-pyrimidine tracts, were incorporated into topologically relaxed minicircles. Negative supercoils were generated, and the resulting topoisomers were resolved by electrophoresis. Using an anti-Z-DNA Fab and an electrophoretic mobility shift assay, Z-DNA was detected in the enhancer-containing minicircles at a superhelix density of -0.05. Fab saturation binding experiments demonstrated that three, independent Z-DNA tracts were stabilized in the supercoiled minicircles. Two other minicircles, each with one of the two alternating purine-pyrimidine tracts, also contained single Z-DNA sites. These results confirm the identities of the Z-DNA-forming sequences within the control region. Moreover, the B-DNA to Z-DNA transitions were detected at superhelix densities observed during normal replication and transcription processes in the SV40 life cycle.