Linker histones affect patterns of digestion of supercoiled plasmids by single-strand-specific nucleases.

2004

Biochemistry

The interaction of linker histone H1 with both linear and superhelical double-stranded DNA has been investigated at low ionic strengths. Gel mobility retardation experiments demonstrate strikingly different behavior for the two forms of DNA. First, the experiments strongly suggest that linker histone binds to superhelical DNA in a negatively cooperative mode. In contrast, binding of linker histone to linear DNA under the conditions employed here shows no cooperativity. Second, binding of linker histone to linear DNA results in aggregation of histone-DNA complexes, even at very low levels of input histone H1. Because H1 has been shown to interact as a monomer, this aggregation is evidence of the divalent character of the linker histone, for without H1's ability to bind to two duplex strands of DNA, aggregation could not occur. Although aggregation can be made to occur with superhelical DNA, it can do so only at near-saturation levels of input histone H1. Finally, in direct competition, linker histone binds to superhelical DNA to the complete exclusion of linear DNA, indicating that the linker histone's function is related to the crossover structures that differentiate superhelical DNA from linear DNA. We develop a model that explains the observed behavior of binding of linker histone to superhelical DNA that is consistent with both the divalent character of the linker histone and the negative cooperativity by which linker histone and superhelical DNA interact.

Section: Discussionmentioning

confidence: 99%

“…Further, it has been established by two kinds of studies that binding of linker histones unwinds supercoiled DNA (20,21). If unwinding to a more weakly binding conformation is a consequence of binding, then that binding must be negatively cooperative.…”

Section: Discussionmentioning

confidence: 99%

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

Ellen

2004

Biochemistry

“…Indeed, a preference of HI for four-way junction DNA (a structure present at the base of cruciforms in supercoiled plasmids) was previously reported from our laboratory (Varga-Weisz et al, 1993. However, such structures should be sensitive to cleavage by single-strand-specific nucleases such as SI or P1; no such cleavages occur in pBR322 at superhelical densities less negative than -0.03 (-13 superhelical turns) (Ivanchenko et al, 1996b;Carnevali et al, 1984). At the same time, the preferential binding of HI is still detectable, even at 1Io -0.012 (Fig.…”

Section: Discussionmentioning

confidence: 99%

Histone H1 preferentially binds to superhelical DNA molecules of higher compaction

Ivanchenko

Zlatanova

1997

Biophysical Journal

In chromatin, the physiological amount of H1 is one molecule per nucleosome or, roughly, one molecule per 200 bp of DNA. We observed that at such a stoichiometry, H1 selectively binds to supercoiled DNA with magnitude of sigma > or = 0.012 (both negative and positive), leaving relaxed, linear, or nicked DNA molecules unbound. When negative and positive DNA topoisomers of varying superhelicity are simultaneously present in the binding mixture, H1 selectively binds to the molecules with highest superhelicity; less supercoiled forms are gradually involved in binding upon increasing the amount of input protein. We explain this topological preference of H1 as the consequence of an increased probability for more than one H1-DNA contact provided by the supercoiling. The existence of simultaneous contacts of H1 with both intertwined DNA strands in the supercoiled DNA molecules is also inferred by topoisomerase relaxation of H1-DNA complexes that had been prefixed with glutaraldehyde.

“…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.…”

mentioning

confidence: 52%

H1 Binding Unwinds

Ivanchenko

Hassan

Journal of Biological Chemistry

et al. 1996

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...