Cells have evolved sophisticated DNA repair systems to correct damaged DNA. However, the human DNA mismatch repair protein Msh2-Msh3 is involved in the process of trinucleotide (CNG) DNA expansion rather than repair. Using purified protein and synthetic DNA substrates, we show that Msh2-Msh3 binds to CAG-hairpin DNA, a prime candidate for an expansion intermediate. CAG-hairpin binding inhibits the ATPase activity of Msh2-Msh3 and alters both nucleotide (ADP and ATP) affinity and binding interfaces between protein and DNA. These changes in Msh2-Msh3 function depend on the presence of A.A mispaired bases in the stem of the hairpin and on the hairpin DNA structure per se. These studies identify critical functional defects in the Msh2-Msh3-CAG hairpin complex that could misdirect the DNA repair process.

The precise positioning of nucleosomes plays a critical role in the regulation of gene expression by modulating the DNA binding activity of trans-acting factors. However, molecular determinants responsible for positioning are not well understood. We examined whether the removal of the core histone tail domains from nucleosomes reconstituted with specific DNA fragments led to alteration of translational positions. Remarkably, we find that removal of tail domains from a nucleosome assembled on a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene results in repositioning of nucleosomes along the DNA, including two related major translational positions that move about 20 bp further upstream with respect to the 5S gene. In a nucleosome reconstituted with a DNA fragment containing the promoter of a Drosophila alcohol dehydrogenase gene, several translational positions shifted by about 10 bp along the DNA upon tail removal. However, the positions of nucleosomes assembled with a DNA fragment known to have one of the highest binding affinities for core histone proteins in the mouse genome were not altered by removal of core histone tail domains. Our data support the notion that the basic tail domains bind to nucleosomal DNA and influence the selection of the translational position of nucleosomes and that once tails are removed movement between translational positions occurs in a facile manner on some sequences. However, the effect of the N-terminal tails on the positioning and movement of a nucleosome appears to be dependent on the DNA sequence such that the contribution of the tails can be masked by very high affinity DNA sequences. Our results suggest a mechanism whereby sequence-dependent nucleosome positioning can be specifically altered by regulated changes in histone tail-DNA interactions in chromatin.

Reconstitution of a DNA fragment containing a 5S RNA gene from Xenopus borealis into a nucleosome greatly restricts binding of the primary 5S transcription factor, TFIIIA. Consistent with transcription experiments using reconstituted templates, removal of the histone tail domains stimulates TFIIIA binding to the 5S nucleosome greater than 100-fold. However, we show that tail removal increases the probability of 5S DNA unwrapping from the core histone surface by only approximately fivefold. Moreover, using site-specific histoneto-DNA cross-linking, we show that TFIIIA binding neither induces nor requires nucleosome movement. Binding studies with COOH-terminal deletion mutants of TFIIIA and 5S nucleosomes reconstituted with native and tailless core histones indicate that the core histone tail domains play a direct role in restricting the binding of TFIIIA. Deletion of only the COOH-terminal transcription activation domain dramatically stimulates TFIIIA binding to the native nucleosome, while further C-terminal deletions or removal of the tail domains does not lead to further increases in TFIIIA binding. We conclude that the unmodified core histone tail domains directly negatively influence TFIIIA binding to the nucleosome in a manner that requires the C-terminal transcription activation domain of TFIIIA. Our data suggest an additional mechanism by which the core histone tail domains regulate the binding of trans-acting factors in chromatin.

We previously reported that reconstituted nucleosomes undergo sequence-dependent translational re-positioning upon removal of the core histone tail domains in physiological conditions, indicating that the tails influence choice of position. We report here that removal of the core histone tail domains increases the exposure of the DNA backbone in nucleosomes to hydroxyl radicals, a non-biased chemical cleavage reagent, indicative of an increase in motility of the DNA on the histone surface. Moreover, we demonstrate that the divalent cations Mg2+ and Ca2+ can replace the role of the tail domains with regard to stabilization of histone-DNA interactions within the nucleosome core and restrict re-positioning of nucleosomes upon tail removal. However, when nucleosomes were incubated with Mg2+ after tail removal, the original distribution of translational positions was not re-attained, indicating that divalent cations increase the energy barrier between translational positions rather than altering the free energy differences between positions. Interestingly, other divalent cations such as Zn2+, Fe2+, Co2+, and Mn2+ had little or no affect on the stability of histone-DNA interactions within tailless nucleosomes. These results support the idea that specific binding sites for Mg2+ and Ca2+ ions exist within the nucleosome and play a critical role in nucleosome stability that is partially redundant with the core histone tail domains.

core histones or to nucleosomes in which these domains are hyperacetylated. The degree to which tail acetylation or removal improves TFIIIA binding cannot be simply explained by a commensurate change in the general accessibility of nucleosomal DNA. In order to investigate the molecular basis of how TFIIIA binds to the nucleosome and to ascertain if binding involves all nine zinc fingers and/or displacement of histone-DNA interactions, we examined the TFIIIA-nucleosome complex by hydroxyl radical footprinting and site-directed protein-DNA cross-linking. Our data reveal that the first six fingers of TFIIIA bind and displace approximately 20 bp of histone-DNA interactions at the periphery of the nucleosome, while binding of fingers 7 to 9 appears to overlap with histone-DNA interactions. Molecular modeling based on these results and the crystal structures of a nucleosome core and a TFIIIA-DNA cocomplex yields a precise picture of the ternary complex and a potentially important intermediate in the transition from naïve chromatin structure to productive polymerase III transcription complex.The assembly of DNA into the basic subunit of chromatin, the nucleosome, severely restricts the activity of most sequence-specific DNA binding factors. This is due to constraints arising from the intimate association of the DNA with histone proteins and the severe DNA bending and changes in helical periodicity that occur upon nucleosome formation (18,26,49). However, nucleosomes are dynamic structures, in rapid equilibrium with states in which the DNA is partially unwound and as accessible to DNA binding proteins as naked DNA (32). Thus, DNA binding transcription factors compete directly with histones for binding to nucleosomal DNA (32). Importantly, Widom and colleagues have demonstrated that this simple competition between histones and trans-acting factors leads to inherent cooperativity in the binding of otherwise unrelated factors and appears to be operative in vivo (28,33).Given the apparent role of chromatin structure in the developmental regulation of the 5S rRNA gene system from Xenopus (1, 3, 48), DNA fragments containing these genes have been useful model systems for investigation of the interplay between the binding of histones and transcription factors to the same DNA (35, 46). DNA fragments containing 5S genes harbor robust nucleosome positioning elements that direct the binding of histones upon reconstitution in vitro (10, 38). Moreover, the initial event in transcriptional activation of the 5S gene family involves binding of the 5S-specific transcription factor IIIA (TFIIIA) to an internal promoter (internal control region) (9). TFIIIA is a nine-zinc-finger protein (4, 27) which binds in a complex fashion along the entire length of the ϳ50-bp internal promoter in three structurally distinct modules (39, 44). A DNA fragment containing a Xenopus borealis somatic-type 5S gene assembles into positioned nucleosomes upon reconstitution with purified core histones (16,35,40). Hydroxyl radical footprinting has shown that str...