H1 Binding Unwinds

Abstract

The preference of the linker histones to bind to superhelical DNA in comparison with linear or relaxed molecules suggests that these proteins might, in turn, change the twist and/or writhe of DNA molecules upon binding. In order to explore such a possibility, we looked for changes in the linking number of plasmid pBR322 caused by H1 binding, using assays that involve nicking and resealing of DNA strands. Two types of enzymes were used, eukaryotic topoisomerase I and prokaryotic DNA ligase. The results revealed that H1 binding causes unwinding of the DNA, with the unwinding angle being approximately 10°. The globular domain of histone H1 is also capable of unwinding DNA, but to a lesser degree.It has been known for a number of years that the linker histones (H1, H5, and their variants) exhibit a preference to bind to superhelical DNA over linear or relaxed DNA (1-3). A reasonable corollary to this observation is that these proteins, in binding to superhelical DNA, might change the twist of the double helix and/or the writhing of the helical axis in space. Consistent with this notion, we have recently shown that titration of superhelical plasmid DNA with increasing amounts of histone H1 changes the patterns of cleavage by single strandspecific nucleases, causing disappearance of preexisting nuclease-sensitive sites at low and moderate H1 levels, followed by the appearance of new sites at high protein levels (4). A possible explanation for the disappearance of preexisting sites is that histone H1 binding absorbs some of the negative superhelical stress in the molecule (unwinds the DNA), so that the decreased stress leads to loss of stress-dependent sensitive sites.Previous work on the binding of histone H1 or H5 to superhelical plasmids has led to contradictory interpretations. BinaStein and Singer (5) interpreted their data as indicating stabilization of preexisting superhelical turns by H1 binding, with no new superhelical turns being created. Stein (6) and Morse and Cantor (7), on the other hand, did not observe stabilization of preexisting superhelical turns. More recently, Sheflin et al. (8) reported that histone H1 was not able to change the linking number of superhelical DNA when assayed in the topoisomerase I-mediated relaxation assay (see below).What is required is a very careful analysis using more than one technique, since the effects might well be small. We decided to utilize both the topoisomerase I-mediated relaxation assay and the ligase-mediated supercoiling assay. The results indicate that linker histones binding unwinds DNA. EXPERIMENTAL PROCEDURESPreparation of Plasmid DNA and Histone H1-Plasmid pBR322 was prepared by CsCl purification and phenol extraction (9). Chicken erythrocyte histone H1 was obtained under nondenaturing conditions (10) and checked for purity by SDS-containing polyacrylamide gel electrophoresis (11). The globular domain of histone H1 was prepared as outlined in Krylov et al. (3). The concentration of the protein stock solutions was estimated by scanning of Coomassie-stained...

Linker Histone Interaction Shows Divalent Character with both Supercoiled and Linear DNA

“…We have shown above and previously that supercoiled DNA binds H1 much more strongly than does linear or relaxed circular DNA. Further, it has been established by two kinds of studies that binding of linker histones unwinds supercoiled DNA ( , ). If unwinding to a more weakly binding conformation is a consequence of binding, then that binding must be negatively cooperative.…”

Linker histones versus HMG1/2: a struggle for dominance?

“…It may also be relevant to note that topological assays have defined linker histones as DNA unwinding proteins, formally unwinding DNA by ϳ10 o per histone molecule bound. (18) This unwinding could be due to bending, similar to that observed with HNF-3 and Ets-1. That linker histones may induce bending may be also expected on the basis of the recent observation that they show a strong preference for binding to DNA which has been bent by modification with the antitumour drug cisplatin.…”

“…Both proteins unwind DNA, albeit to a different angle. (17,18) Although in general the binding is sequence non-specific, a number of cases have been reported where both H1 (19,20) (for earlier review, see ref. 21) and HMG1 (22) possess some sequence specificity.…”

Temperature and osmotic stress dependence of the thermodynamics for binding linker histone H1 0 , Its carboxyl domain (H1 0 -C) or globular domain (H1 0 -G) to B-DNA

“…Our previous CD work seemed to suggest that any DNA structural changes accompanying the binding of H1 0 -G are not observable. However, ligation assays done by Maria et al revealed that binding of H1 globular domains causes some unwinding of superhelical DNA [30]. …”