High mobility group protein 1 preferentially conserves torsion in negatively supercoiled DNA

Abstract

HMG 1 is known to bind to a variety of DNAs and to unwind nicked and closed circular DNA. We now report evidence that it has a significantly higher unwinding angle on negatively supercoiled DNA than on the other torsional forms. The degree of unwinding observed on nicked circular DNA depends on the purity of the HMG 1 preparation used. HMG 1 from CM-Sephadex has an unwinding angle of 28.8.degree., compared to 7.2.degree. for the purer preparation obtained from Mono S, suggesting that contaminating strand-separating activity is removed by the additional purification step. The subsequent studies on closed circular forms of DNA were all performed using the purer HMG 1. After preincubation of highly negatively supercoiled DNA (.sigma. = -0.040) with HMG 1, the DNA-protein mixture was relaxed with Escherichia coli topoisomerase I. At molar ratios of less than 100:1 (HMG 1 to DNA), negatively supercoiled DNA displays a dose-dependent change in the linking number, indicating an unwinding angle of 57.6.degree.. HMG 1 protects 50% of highly negatively supercoiled DNA from E. coli topoisomerase I at a molar ratio of 100:1, and protects all supercoils at a molar ratio of 200:1, indicating saturation of the DMA at this concentration. HMG 1 also protects highly negatively supercoiled DNA from calf thymus topoisomerase I, with an apparent unwinding angle of 57.6.degree.. Moderately negatively supercoiled DNA (.sigma. = -0.018), but not moderately positively supercoiled DNA (.sigma. = +0.011), competes for the protective effect of HMG 1 on highly negatively supercoiled DNA. Preincubation of negatively supercoiled DNA with HMG 1 also protects it from mung bean nuclease cleavage, but this protection is not complete. The data suggest that HMG 1 preferentially interacts with negatively supercoiled DNA, absorbing its tension, which prevents formation of supercoil-dependent secondary structures. The role HMG 1 plays in the regulation of transcription and DNA replication thus may be related to its ability to preferentially relax negatively supercoiled domains in chromatin. This general mode of action of HMG 1 would permit conservation of higher order DNA structures and could facilitate the binding of sequence-specific regulatory factors.