Histone-DNA binding free energy cannot be measured in dilution-driven dissociation experiments

Despite decades of study on nucleosomes, there has been no experimental determination of the free energy of association between histones and DNA. Instead, only the relative free energy of association of the histone octamer for differing DNA sequences has been available. Recently, a method was developed based on quantitative analysis of nucleosome dissociation in dilution experiments that provides a simple practical measure of nucleosome stability. Solution conditions were found in which nucleosome dissociation driven by dilution fit well to a simple model involving a noncooperative nucleosome assembly/disassembly equilibrium, suggesting that this approach might allow absolute equilibrium affinity of the histone octamer for DNA to be measured. Here, we show that the nucleosome assembly/disassembly process is not strictly reversible in these solution conditions, implying that equilibrium affinities cannot be obtained from these measurements. Increases in [NaCl] or temperature, commonly employed to suppress kinetic bottlenecks in nucleosome assembly, lead to cooperative behavior that cannot be interpreted with the simple assembly/disassembly equilibrium model. We conclude that the dilution experiments provide useful measures of kinetic but not equilibrium stability. Kinetic stability is of practical importance: it may govern nucleosome function in vivo, and it may (but need not) parallel absolute thermodynamic stability.     

hMSH2-hMSH6 forms a hydrolysis-independent sliding clamp on mismatched DNA

Mismatch recognition by the human MutS homologs hMSH2-hMSH6 is regulated by adenosine nucleotide binding, supporting the hypothesis that it functions as a molecular switch. Here we show that ATP-induced release of hMSH2-hMSH6 from mismatched DNA is prevented if the ends are blocked or if the DNA is circular. We demonstrate that mismmatched DNA provokes ADP-->ATP exchange, resulting in a discernible conformational transition that converts hMSH2-hMSH6 into a sliding clamp capable of hydrolysis-independent diffusion along the DNA backbone. Our results support a model for bidirectional mismatch repair in which stochastic loading of multiple ATP-bound hMSH2-hMSH6 sliding clamps onto mismatch-containing DNA leads to activation of the repair machinery and/or other signaling effectors similar to G protein switches.     

CENP-A and H3 Nucleosomes Display a Similar Stability to Force-Mediated Disassembly

Centromere-specific nucleosomes are a central feature of the kinetochore complex during mitosis, in which microtubules exert pulling and pushing forces upon the centromere. CENP-A nucleosomes have been assumed to be structurally unique, thereby providing resilience under tension relative to their H3 canonical counterparts. Here, we directly test this hypothesis by subjecting CENP-A and H3 octameric nucleosomes, assembled on random or on centromeric DNA sequences, to varying amounts of applied force by using single-molecule magnetic tweezers. We monitor individual disassembly events of CENP-A and H3 nucleosomes. Regardless of the DNA sequence, the force-mediated disassembly experiments for CENP-A and H3 nucleosomes demonstrate similar rupture forces, life time residency and disassembly steps. From these experiments, we conclude that CENP-A does not, by itself, contribute unique structural features to the nucleosome that lead to a significant resistance against force-mediated disruption. The data present insights into the mechanistic basis for how CENP-A nucleosomes might contribute to the structural foundation of the centromere in vivo.     

Formation of hMSH4-hMSH5 heterocomplex is a prerequisite for subsequent GPS2 recruitment

Increasing evidence suggests that components of the DNA mismatch repair (MMR) pathway play multifunctional roles beyond the scope of mismatch correction, including the modulation of cellular responses to DNA damage and homologous recombination. The heterocomplex consisting of MutS homologous proteins, hMSH4 and hMSH5, is believed to play essential roles in meiotic DNA repair particularly during the process of meiotic homologous recombination (HR). In order to gain a better understanding of the mechanistic basis underlying the roles of these two human MutS proteins, we have identified G-protein pathway suppressor 2 (GPS2) (i.e., an integral component of a deacetylase complex) as an interacting protein partner specifically for the hMSH4-hMSH5 heterocomplex. The interaction with GPS2 is entirely dependent on the physical association between hMSH4 and hMSH5, as disruption of the interaction between hMSH4 and hMSH5 completely abolishes GPS2 recruitment. Our analysis further indicates that the association with GPS2 is mediated through the interface of hMSH4-hMSH5 complex and the N-terminal region of GPS2. Moreover, these three proteins interact in human cells, and analysis of microarray data suggested a coordinated expression pattern of these genes during the onset of meiosis. Together, the results of our present study suggest that the GPS2-associated deacetylase complex might function in concert with hMSH4-hMSH5 during the process of homologous recombination.     

Recent advances in single molecule studies of nucleosomes

As the fundamental packing units of DNA in eukaryotes, nucleosomes play a central role in governing DNA accessibility in a variety of cellular processes. Our understanding of the mechanisms underlying this complex regulation has been aided by unique structural and dynamic perspectives offered by single molecule techniques. Recent years have witnessed remarkable advances achieved using these techniques, including the generation of a detailed histone-DNA energy landscape, elucidation of nucleosome disassembly processes, and real-time monitoring of molecular motors interacting with nucleosomes. These and other highlights of single molecule nucleosome studies will be discussed in this review.     

The effect of O6-methylguanine DNA adducts on the adenosine nucleotide switch functions of hMSH2-hMSH6 and hMSH2-hMSH3

The human homologs of prokaryotic mismatch repair have been shown to mediate the toxicity of certain DNA damaging agents; cells deficient in the mismatch repair pathway exhibit resistance to the killing effects of several of these agents. Although previous studies have suggested that the human MutS homologs, hMSH2-hMSH6, bind to DNA containing a variety of DNA adducts, as well as mispaired nucleotides, a number of studies have suggested that DNA binding does not correlate with repair activity. In contrast, the ability to process adenosine nucleotides by MutS homologs appears to be fundamentally linked to repair activity. In this study, oligonucleotides containing a single well defined O(6)-methylguanine adduct were used to examine the extent of lesion-provoked DNA binding, single-step ADP --> ATP exchange, and steady-state ATPase activity by hMSH2-hMSH3 and hMSH2-hMSH6 heterodimers. Interestingly, O(6)-methylguanine lesions when paired with either a C or T were found to stimulate ADP --> ATP exchange, as well as the ATPase activity of purified hMSH2-hMSH6, whereas there was no significant stimulation of hMSH2-hMSH3. These results suggest that O(6)-methylguanine uniquely activates the molecular switch functions of hMSH2-hMSH6.     

hMSH4-hMSH5 recognizes Holliday Junctions and forms a meiosis-specific sliding clamp that embraces homologous chromosomes

Five MutS homologs (MSH), which form three heterodimeric protein complexes, have been identified in eukaryotes. While the human hMSH2-hMSH3 and hMSH2-hMSH6 heterodimers operate primarily in mitotic mismatch repair (MMR), the biochemical function(s) of the meiosis-specific hMSH4-hMSH5 heterodimer is unknown. Here, we demonstrate that purified hMSH4-hMSH5 binds uniquely to Holliday Junctions. Holliday Junctions stimulate the hMSH4-hMSH5 ATP hydrolysis (ATPase) activity, which is controlled by Holliday Junction-provoked ADP-->ATP exchange. ATP binding by hMSH4-hMSH5 induces the formation of a hydrolysis-independent sliding clamp that dissociates from the Holliday Junction crossover region, embracing two homologous duplex DNA arms. Fundamental differences between hMSH2-hMSH6 and hMSH4-hMSH5 Holliday Junction recognition are detailed. Our results support the attractive possibility that hMSH4-hMSH5 stabilizes and preserves a meiotic bimolecular double-strand break repair (DSBR) intermediate.     

Micromanipulation studies of chromatin fibers in Xenopus egg extracts reveal ATP-dependent chromatin assembly dynamics

We have studied assembly of chromatin using Xenopus egg extracts and single DNA molecules held at constant tension by using magnetic tweezers. In the absence of ATP, interphase extracts were able to assemble chromatin against DNA tensions of up to 3.5 piconewtons (pN). We observed force-induced disassembly and opening-closing fluctuations, indicating our experiments were in mechanochemical equilibrium. Roughly 50-nm (150-base pair) lengthening events dominated force-driven disassembly, suggesting that the assembled fibers are chiefly composed of nucleosomes. The ATP-depleted reaction was able to do mechanical work of 27 kcal/mol per 50 nm step, which provides an estimate of the free energy difference between core histone octamers on and off DNA. Addition of ATP led to highly dynamic behavior with time courses exhibiting processive runs of assembly and disassembly not observed in the ATP-depleted case. With ATP present, application of forces of 2 pN led to nearly complete fiber disassembly. Our study suggests that ATP hydrolysis plays a major role in nucleosome rearrangement and removal and that chromatin in vivo may be subject to highly dynamic assembly and disassembly processes that are modulated by DNA tension.     

Human MSH2 (hMSH2) protein controls ATP processing by hMSH2-hMSH6

The mechanics of hMSH2-hMSH6 ATP binding and hydrolysis are critical to several proposed mechanisms for mismatch repair (MMR), which in turn rely on the detailed coordination of ATP processing between the individual hMSH2 and hMSH6 subunits. Here we show that hMSH2-hMSH6 is strictly controlled by hMSH2 and magnesium in a complex with ADP (hMSH2(magnesium-ADP)-hMSH6). Destabilization of magnesium results in ADP release from hMSH2 that allows high affinity ATP binding by hMSH6, which then enhances ATP binding by hMSH2. Both subunits must be ATP-bound to efficiently form a stable hMSH2-hMSH6 hydrolysis-independent sliding clamp required for MMR. In the presence of magnesium, the ATP-bound sliding clamps remain on the DNA for ～8 min. These results suggest a precise stepwise kinetic mechanism for hMSH2-hMSH6 functions that appears to mimic G protein switches, severely constrains models for MMR, and may partially explain the MSH2 allele frequency in Lynch syndrome or hereditary nonpolyposis colorectal cancer.     

Bcl2 impedes DNA mismatch repair by directly regulating the hMSH2-hMSH6 heterodimeric complex

Bcl2 has been reported to suppress DNA mismatch repair (MMR) with promotion of mutagenesis, but the mechanism(s) is not fully understood. MutSalpha is the hMSH2-hMSH6 heterodimer that primarily functions to correct mutations that escape the proofreading activity of DNA polymerase. Here we have discovered that Bcl2 potently suppresses MMR in association with decreased MutSalpha activity and increased mutagenesis. Exposure of cells to nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone results in accumulation of Bcl2 in the nucleus, which interacts with hMSH6 but not hMSH2 via its BH4 domain. Deletion of the BH4 domain from Bcl2 abrogates the ability of Bcl2 to interact with hMSH6 and is associated with enhanced MMR efficiency and decreased mutation frequency. Overexpression of Bcl2 reduces formation of the hMSH2-hMSH6 complex in cells, and purified Bcl2 protein directly disrupts the hMSH2-hMSH6 complex and suppresses MMR in vitro. Importantly, depletion of endogenous Bcl2 by RNA interference enhances formation of the hMSH2-hMSH6 complex in association with increased MMR and decreased mutagenesis. Thus, Bcl2 suppression of MMR may occur in a novel mechanism by directly regulating the heterodimeric hMSH2-hMSH6 complex, which potentially contributes to genetic instability and carcinogenesis.     

Sin mutations alter inherent nucleosome mobility

Previous studies have identified sin mutations that alleviate the requirement for the yeast SWI/SNF chromatin remodelling complex, which include point changes in the yeast genes encoding core histones. Here we characterise the biochemical properties of nucleosomes bearing these mutations. We find that sin mutant nucleosomes have a high inherent thermal mobility. As the SWI/SNF complex can alter nucleosome positioning, the higher mobility of sin mutant nucleosomes provides a means by which sin mutations may substitute for SWI/SNF function. The location of sin mutations also provides a new opportunity for insights into the mechanism for nucleosome mobilisation. We find that both mutations altering histone DNA contacts at the nucleosome dyad and mutations in the dimer-tetramer interface influence nucleosome mobility. Furthermore, incorporation of H2A.Z into nucleosomes, which also alters dimer-tetramer interactions, affects nucleosome mobility. Thus, variation of histone sequence or subtype provides a means by which eukaryotes may regulate access to chromatin through alterations to nucleosome mobility.