Ribonucleotide-induced helical alteration in DNA prevents nucleosome formation.

Abstract

Several polynucleotides that assume an Aform helical structure in solution are unable to form nucleosomes. We attempted to establish a relationship between the ease of the A-form -+ B-form helix transition and ease of nucleosome formation by reconstituting nucleosomes using ribosubstituted DNA containing various levels of ribonucleotides. Instead we discovered that, when riboadenosine is substituted for deoxyriboadenosine, even one ribonucleotide per 125 base pairs of DNA reduces nucleosome formation and that DNA containing >5% ribonucleotide is completely unable to form nucleosomes. Ribosubstituted DNA restriction fragments exhibited altered mobility on native 6% polyacrylamide gels, indicating an altered helical structure (probably bending).The effects on both nucleosome formation and gel mobility are nucleotide specific and are correlated, being greatest for riboadenosine and decreasing in the order riboadenosine > riboguanosine > ribocytosine. The results are consistent with the hypothesis that the rate of nucleosome formation can be drastically reduced by isolated local perturbations, such as kinking or bending, in the helical structure of DNA.We are interested in the manner by which DNA helix structure modulates its own mode of packaging by histones in chromatin. In some cases the effects on packaging have obvious physiological implications. For example, the positioning of artificial nucleosomes on Escherichia coli lac operator DNA (1) constrains the DNA in such a way that the repressor binding surface of the DNA always faces outward to bind repressor (2). Eukaryotic examples include the specific positioning of nucleosomes on 5S gene DNA (3) so as to facilitate binding by transcription factor IIIA (4) and the avoidance by nucleosomes of poly(dA)-poly(dT) tracts in DNA (5, 6), which probably accounts for the promoteractivating properties of this sequence in chromatin (7).It now appears that the sequence-specific positioning of nucleosomes and the sidedness with which DNA binds the histone core may be explained by the tendency of certain short sequence elements (8) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Nucleosome assembly of simian virus 40 DNA in a mammalian cell extract.

“…Mg2+-induced conformational alterations of SV40 DNA lead to nuclease sensitivity of several distinct sequences, including its control elements (31). It is evident that DNA sequences can determine the position of nucleosomes (14,51), and their local conformations might influence the extent of assembly (29,30,47,48 (16,60), and the concentration and type of counterions in solution (27,49,63). To characterize the effect of Mg2+ on the global conformation of SV40 DNA, we relaxed supercoiled SV40 DNA with purified topoisomerase I in the presence or absence of Mg2+ and analyzed the resulting species by electrophoresis in Mg2+-free buffer in the presence or absence of chloroquine.…”

Processing ribonucleotides incorporated during eukaryotic DNA replication

“…The formation of these alternative base pairs in DNA may have functional importance as it expands the structural repertoire of duplex DNA, potentially influencing protein-DNA interactions and processes that include replication, repair and recombination ( 87 and references therein). Chromatin assembly and reassembly as well as nucleosome positioning may also be affected by ribonucleotides, as nucleosome binding to DNA in vitro is reduced when ribonucleotides are embedded in DNA 88,89 . Altogether, the structural perturbations caused by ribonucleotides incorporated into DNA have the potential to impact multiple important fundamental biological processes, including DNA replication, transcription, DNA repair, recombination and chromosome segregation.…”

Ribonucleotides in DNA: Origins, repair and consequences

“…They may influence telomere function, because they are incorporated by telomerase [41], their presence in DNA reduces DNA binding by the telomere binding protein Pot1 [94], loss of RNase H2 in yeast is associated with increased expression of genes involved in telomere maintenance [65], and the yeast RNH201 gene was recovered in two screens [80,95] for genes that affect telomere length. Ribonucleotides in DNA reduce nucleosome binding [96,97], possibly influencing histone re-loading behind the replication fork and/or nucleosome positioning or dynamics. This idea could possibly be related to the fact that Pol ε , which incorporates ribonucleotides into the nascent leading strand, is involved in gene silencing and one of its non-catalytic subunits is involved in chromatin remodeling (reviewed in [98]).…”