DNA-Protein Cross-Link Formation in Nucleosome Core Particles Treated with Methyl Methanesulfonate.
ABSTRACT: N7-Methyl-2'-deoxyguanosine (MdG) is the major damage product in DNA produced by methylating agents, but it often thought to be nontoxic and nonmutagenic. MdG is chemically unstable. An abasic site (AP) is the major product produced from MdG under physiologically relevant conditions. AP formation is frequently considered to be responsible for the cytotoxic effects of MdG, but the reaction is suppressed in nucleosome core particles (NCPs). Recently, it was discovered that histone proteins form reversible DNA-protein cross-links (DPCs) with MdG in reconstituted NCPs, as well as in methylmethanesulfonate (MMS) treated cells. In this study, the formation and reactivity of MdG in MMS treated NCPs was examined at single nucleotide resolution. Sequences consisting of three or more consecutive dGs are more reactive with MMS. The efficiency and selectivity of MdG formation by MMS is largely unaffected within a NCP, although reactivity at several dGs is ?1.5-2.5-fold higher in NCPs. DPC formation from MdG (DPCMdG) predominates over AP at all positions within the NCP. With few exceptions, DPCMdG yield is strongly dependent upon the accessibility of the major groove containing MdG to lysine-rich histone N-terminal tails. These data indicate that histone-MdG DPC formation will depend upon DNA sequence and translational position within an NCP.
Project description:Monofunctional alkylating agents preferentially react at the N7 position of 2'-deoxyguanosine in duplex DNA. Methylated DNA, such as that produced by methyl methanesulfonate (MMS) and temozolomide, exists for days in organisms. The predominant consequence of N7-methyl-2'-deoxyguanosine (MdG) is widely believed to be abasic site (AP) formation via hydrolysis, a process that is slow in free DNA. Examination of MdG reactivity within nucleosome core particles (NCPs) provided two general observations. MdG depurination rate constants are reduced in NCPs compared with when the identical DNA sequence is free in solution. The magnitude of the decrease correlates with proximity to the positively charged histone tails, and experiments in NCPs containing histone variants reveal that positively charged amino acids are responsible for the decreased rate of abasic site formation from MdG. In addition, the lysine-rich histone tails form DNA-protein cross-links (DPCs) with MdG. Cross-link formation is reversible and is ascribed to nucleophilic attack at the C8 position of MdG. DPC and retarded abasic site formation are observed in NCPs randomly damaged by MMS, indicating that these are general processes. Histone-MdG cross-links were also detected by mass spectrometry in chromatin isolated from V79 Chinese hamster lung cells treated with MMS. The formation of DPCs following damage by a monofunctional alkylating agent has not been reported previously. These observations reveal the possibility that such DPCs may contribute to the cytotoxicity of monofunctional alkylating agents, such as MMS, N-methyl-N-nitrosourea, and temozolomide.
Project description:N3-Methyl-2'-deoxyadenosine (MdA) is the major dA methylation product in duplex DNA. MdA blocks DNA replication and undergoes depurination at significantly higher rates than the native nucleotide from which it is derived. Recent reports on the effects of the nucleosome core particle (NCP) environment on the reactivity of N7-methyl-2'-deoxyguanosine (MdG) inspired this investigation concerning the reactivity of MdA in NCPs. NCPs containing MdA at selected positions were produced using a strategy in which the minor groove binding Me-Lex molecule serves as a sequence specific methylating agent. Hydrolysis of the glycosidic bond in MdA to form abasic sites (AP) is suppressed in a NCP. Experiments using histone variants indicate that the proximal, highly basic N-terminal tails are partially responsible for the decreased depurination rate constant. MdA also forms cross-links with histone proteins. The levels of MdA-histone DNA-protein cross-links (DPCMdA) decrease significantly over time and are replaced by those involving AP. The time dependent decrease in DPCMdA is attributed to the reversibility of their formation and the relatively rapid rate of AP formation from MdA. Overall, MdA reactivity in NCPs qualitatively resembles that of MdG.
Project description:Duplex DNA containing an apurinic/apyrimidinic (AP) lesion undergoes cleavage significantly more rapidly in nucleosome core particles (NCPs) than it does when free. The mechanism of AP cleavage within NCPs was studied through independently generating lesions within them. AP mediated DNA cleavage within NCPs is initiated by DNA-protein cross-link (DPC(un)) formation followed by ?-elimination to give DPCs containing cleaved DNA (DPC(cl)). Hydrolysis of DPC(cl) produces a DNA single strand break (SSB). C2-dideuteration of AP showed that deprotonation from this position is involved in the rate-determining step. Experiments utilizing NCPs containing mutated histone H4 proteins indicated that lysine residues in the amino terminal tail are involved in both DPC formation and ?-elimination steps. Lysines 16 and 20 seem to play a greater role in reacting with AP at superhelical location 1.5, but other amino acids (e.g., lysines 5, 8, and 12) compensate in their absence. The mechanism of rapid double strand breaks in bistranded, clustered AP lesions was studied by independently preparing reaction intermediates within model NCPs. A single strand break on one strand enhances the cleavage of a proximal AP on the opposite strand.
Project description:8-Oxo-7,8-dihydro-2'-deoxyguanosine(8-oxodGuo) is a common primary product of cellular oxidative DNA damage. 8-OxodGuo is more readily oxidized than 2'-deoxyguanosine (dG); a two-electron oxidation generates a highly reactive intermediate (OGox), which forms covalent adducts with nucleophiles, including OH-, free amines, and the side chains of amino acids such as lysine. We determined here that K3Fe(CN)6 oxidation of 8-oxodGuo in nucleosome core particles (NCPs) produces high yields, quantitative (i.e., 100%) in some cases, of DNA-protein cross-links (DPCs). The efficiency of DPC formation was closely related to 8-oxodGuo base pairing and location within the NCP and was only slightly decreased by adding the DNA-protective polyamine spermine to the system. Using NCPs that contained histone mutants, we determined that DPCs result predominantly from OGox trapping by the N-terminal histone amine. The DPCs were stable under physiological conditions and therefore could have important biological consequences. For instance, the essentially quantitative yield of DPCs at some positions within NCPs would reduce the yield of the mutagenic DNA lesions spiroiminodihydantoin and guanidinohydantoin produced from the common intermediate OGox, which in turn would affect mutation signatures of oxidative stress in a position-dependent manner. In summary, our findings indicate that site-specific incorporation of 8-oxodGuo into NCPs, followed by its oxidation, leads to DPCs with an efficiency depending on 8-oxodGuo location and orientation. Given that 8-oxodGuo formation is widespread in genomic DNA and that DPC formation is highly efficient, DPCs may occur in eukaryotic cells and may affect several important biological processes.
Project description:The nucleosome core particle (NCP) is the basic building block of chromatin. Nucleosome-nucleosome interactions are instrumental in chromatin compaction, and understanding NCP self-assembly is important for understanding chromatin structure and dynamics. Recombinant NCPs aggregated by multivalent cations form various ordered phases that can be studied by x-ray diffraction (small-angle x-ray scattering). In this work, the effects on the supramolecular structure of aggregated NCPs due to lysine histone H4 tail acetylations, histone H2A mutations (neutralizing the acidic patch of the histone octamer), and the removal of histone tails were investigated. The formation of ordered mainly hexagonal columnar NCP phases is in agreement with earlier studies; however, the highly homogeneous recombinant NCP systems used in this work display a more compact packing. The long-range order of the NCP columnar phase was found to be abolished or reduced by acetylation of the H4 tails, acidic patch neutralization, and removal of the H3 and H2B tails. Loss of nucleosome stacking upon removal of the H3 tails in combination with other tails was observed. In the absence of the H2A tails, the formation of an unknown highly ordered phase was observed.
Project description:An abasic (AP) site is a ubiquitous DNA lesion that is produced via several cellular processes. Although AP sites are cytotoxic and mutagenic, cells are protected from them by different DNA damage tolerance and repair pathways, including base excision repair (BER). AP lesions are alkali-labile, but the half-life for strand scission is several weeks in free DNA at around neutral pH. The AP lifetime is reduced ?100-fold in nucleosome core particles (NCPs) because the histone proteins promote strand scission. The reactivity of other DNA lesions to BER enzymes and exogenous reagents is highly dependent upon rotational positioning within the NCP. We examined strand scission at AP sites as a function of rotational position over approximately one helical turn of DNA. The rate constant for strand scission at AP varies ?4-fold, a range of reactivity much smaller than that observed for processes that involve reaction with diffusible reagents in solution. In addition, the change in rate constant does not exhibit an obvious pattern with respect to rotational position. The small dependence of reactivity on rotational position is attributed to interactions with histone proteins. A molecular model based upon NCP X-ray crystal structures indicates that histone protein tails access AP sites via the major or minor groove and are therefore not limited to regions where one particular groove is exposed to solvent. Determining the roles of individual proteins is difficult because of the unstructured nature of the histone tails and the chemical mechanism, which involves reversible Schiff base formation, followed by irreversible elimination.
Project description:In the eukaryotic cell nucleus, DNA exists as chromatin, a compact but dynamic complex with histone proteins. The first level of DNA organization is the linear array of nucleosome core particles (NCPs). The NCP is a well-defined complex of 147 bp DNA with an octamer of histones. Interactions between NCPs are of paramount importance for higher levels of chromatin compaction. The polyelectrolyte nature of the NCP implies that nucleosome-nucleosome interactions must exhibit a great influence from both the ionic environment as well as the positively charged and highly flexible N-terminal histone tails, protruding out from the NCP. The large size of the system precludes a modelling analysis of chromatin at an all-atom level and calls for coarse-grained approximations. Here, a model of the NCP that include the globular histone core and the flexible histone tails described by one particle per each amino acid and taking into account their net charge is proposed. DNA wrapped around the histone core was approximated at the level of two base pairs represented by one bead (bases and sugar) plus four beads of charged phosphate groups. Computer simulations, using a Langevin thermostat, in a dielectric continuum with explicit monovalent (K(+)), divalent (Mg(2+)) or trivalent (Co(NH(3))(6) (3+)) cations were performed for systems with one or ten NCPs. Increase of the counterion charge results in a switch from repulsive NCP-NCP interaction in the presence of K(+), to partial aggregation with Mg(2+) and to strong mutual attraction of all 10 NCPs in the presence of CoHex(3+). The new model reproduced experimental results and the structure of the NCP-NCP contacts is in agreement with available data. Cation screening, ion-ion correlations and tail bridging contribute to the NCP-NCP attraction and the new NCP model accounts for these interactions.
Project description:The nucleosome core particle (NCP) is the fundamental building block of chromatin which compacts approximately 146 bp of DNA around a core histone protein octamer. The effects of NCP packaging on long-range DNA charge transport reactions have not been adequately assessed to date. Here we study DNA hole transport reactions in a 157 bp DNA duplex (AQ-157TG) incorporating multiple repeats of the DNA TG-motif, a strong NCP positioning sequence and a covalently attached Anthraquinone photooxidant. Following a thorough biophysical characterization of the structure of AQ-157TG NCPs by Exonuclease III and hydroxyl radical footprinting, we compared the dynamics of DNA charge transport in ultraviolet-irradiated free and NCP-incorporated AQ-157TG. Compaction into a NCP changes the charge transport dynamics in AQ-157TG drastically. Not only is the overall yield of oxidative lesions decreased in the NCPs, but the preferred sites of oxidative damage change as well. This NCP-dependent attenuation of DNA charge transport is attributed to DNA-protein interactions involving the folded histone core since removal of the histone tails did not perturb the charge transport dynamics in AQ-157TG NCPs.
Project description:Coarse-grained Langevin molecular dynamics computer simulations were conducted for systems that mimic solutions of nucleosome core particles (NCPs). The NCP was modeled as a negatively charged spherical particle representing the complex of DNA and the globular part of the histones combined with attached strings of connected charged beads modeling the histone tails. The size, charge, and distribution of the tails relative to the core were built to match real NCPs. Three models of NCPs were constructed to represent different extents of covalent modification on the histone tails: (nonmodified) recombinant (rNCP), acetylated (aNCP), and acetylated and phosphorylated (paNCP). The simulation cell contained 10 NCPs in a dielectric continuum with explicit mobile counterions and added salt. The NCP-NCP interaction is decisively dependent on the modification state of the histone tails and on salt conditions. Increasing the monovalent salt concentration (KCl) from salt-free to physiological concentration leads to NCP aggregation in solution for rNCP, whereas NCP associates are observed only occasionally in the system of aNCPs. In the presence of divalent salt (Mg(2+)), rNCPs form dense stable aggregates, whereas aNCPs form aggregates less frequently. Aggregates are formed via histone-tail bridging and accumulation of counterions in the regions of NCP-NCP contacts. The paNCPs do not show NCP-NCP interaction upon addition of KCl or in the presence of Mg(2+). Simulations for systems with a gradual substitution of K(+) for Mg(2+), to mimic the Mg(2+) titration of an NCP solution, were performed. The rNCP system showed stronger aggregation that occurred at lower concentrations of added Mg(2+), compared to the aNCP system. Additional molecular dynamics simulations performed with a single NCP in the simulation cell showed that detachment of the tails from the NCP core was modest under a wide range of salt concentrations. This implies that salt-induced tail dissociation of the histone tails from the globular NCP is not in itself a major factor in NCP-NCP aggregation. The approximation of coarse-graining, with respect to the description of the NCP as a sphere with uniform charge distribution, was tested in control simulations. A more detailed description of the NCP did not change the main features of the results. Overall, the results of this work are in agreement with experimental data reported for NCP solutions and for chromatin arrays.
Project description:Nucleosomes, the basic unit of chromatin, are repetitively spaced along DNA and regulate genome expression and maintenance. The long linear chromatin molecule is extensively condensed to fit DNA inside the nucleus. How distant nucleosomes interact to build tertiary chromatin structure remains elusive. In this study, we used cryo-EM to structurally characterize different states of long range nucleosome core particle (NCP) interactions. Our structures show that NCP pairs can adopt multiple conformations, but, commonly, two NCPs are oriented with the histone octamers facing each other. In this conformation, the dyad of both nucleosome core particles is facing the same direction, however, the NCPs are laterally shifted and tilted. The histone octamer surface and histone tails in trans NCP pairs remain accessible to regulatory proteins. The overall conformational flexibility of the NCP pair suggests that chromatin tertiary structure is dynamic and allows access of various chromatin modifying machineries to nucleosomes.