The Dnmt1 DNA-(cytosine-C5)-methyltransferase methylates DNA processively with high preference for hemimethylated target sites

In the cell, Dnmt1 is the major enzyme in maintenance of the pattern of DNA methylation after DNA replication. Evidence suggests that the protein is located at the replication fork, where it could directly modify nascent DNA immediately after replication. To elucidate the potential mechanism of this process, we investigate the processivity of DNA methylation and accuracy of copying an existing pattern of methylation in this study using purified Dnmt1 and hemimethylated substrate DNA. We demonstrate that Dnmt1 methylates a hemimethylated 958-mer substrate in a highly processive reaction. Fully methylated and unmethylated CG sites do not inhibit processive methylation of the DNA. Extending previous work, we show that unmethylated sites embedded in a hemimethylated context are modified at an approximately 24-fold reduced rate, which demonstrates that the enzyme accurately copies existing patterns of methylation. Completely unmodified DNA is methylated even more slowly due to an allosteric activation of Dnmt1 by methylcytosine-containing DNA. Interestingly, Dnmt1 is not able to methylate hemimethylated CG sites on different strands of the DNA in a processive manner, indicating that Dnmt1 keeps its orientation with respect to the DNA while methylating the CG sites on one strand of the DNA.     

Accuracy of DNA methylation pattern preservation by the Dnmt1 methyltransferase

DNA methyltransferase 1 (Dnmt1) has a central role in copying the pattern of DNA methylation after replication which is one manifestation of epigenetic inheritance. With oligonculeotide substrates we show that mouse Dnmt1 has a 30- to 40-fold preference for hemimethylated DNA that is almost lost after addition of fully methylated oligonucleotides. Using long hemimethylated DNA substrates that carry defined methylation patterns and bisulfite analysis of the methylation reaction products, we show a 15-fold preference for hemimethylated CG sites. Dnmt1 moves along the DNA in a random walk methylating hemimethylated substrates with high processivity (>50 sites are visited on average which corresponds to linear diffusion over 6000 bp). The frequency of skipping sites is very low (<0.3%) and there is no detectable flanking sequence preference. CGCTC sites tend to terminate the processive methylation of DNA by Dnmt1. Unmethylated DNA is modified non-processively with a preference for methylation at CCGG sites. We simulate the propagation of methylation patterns using a stochastic model with the specificity of Dnmt1 observed here and conclude that either methylation of several sites is required to propagate the methylation information over several cellular generations or additional epigenetic information must be used.     

Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase

DNA methyltransferase Dnmt1 ensures clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during replication. Dnmt1 is targeted to replication foci, interacts with PCNA, and favors methylating the hemimethylated form of CpG sites. To understand the underlying mechanism of its maintenance function, we purified recombinant forms of full-length Dnmt1, a truncated form of Dnmt1-(291-1620) lacking the binding sites for PCNA and DNA and examined their processivity using a series of long unmethylated and hemimethylated DNA substrates. Direct analysis of methylation patterns using bisulfite-sequencing and hairpin-PCR techniques demonstrated that full-length Dnmt1 methylates hemimethylated DNA with high processivity and a fidelity of over 95%, but unmethylated DNA with much less processivity. The truncated form of Dnmt1 showed identical properties to full-length Dnmt1 indicating that the N-terminal 290-amino acid residue region of Dnmt1 is not required for preferential activity toward hemimethylated sites or for processivity of the enzyme. Remarkably, our analyses also revealed that Dnmt1 methylates hemimethylated CpG sites on one strand of double-stranded DNA during a single processive run. Our findings suggest that these inherent enzymatic properties of Dnmt1 play an essential role in the faithful and efficient maintenance of methylation patterns in the mammalian genome.     

Xeroderma pigmentosum complementation group A protein acts as a processivity factor

We have previously shown that endonucleases present in a protein complex, which has specificity for cyclobutane pyrimidine dimers, locate sites of damage in DNA by a processive mechanism of action in normal human lymphoblastoid cells. In contrast, the endonucleases present in this complex from xeroderma pigmentosum complementation group A (XPA) cells locate damage sites by a distributive or significantly less processive mechanism. Since the XPA protein has been shown to be responsible for the DNA repair defect in XPA cells, this protein was examined for involvement in the mechanism of target site location of these endonucleases. A recombinant XPA protein, produced by expression of the normal XPA cDNA in E. coli, was isolated and purified. The results show that the recombinant XPA protein was able to correct the defect in ability of the XPA endonucleases to act by a processive mechanism of action on UVC irradiated DNA. These studies indicate that the XPA protein, in addition to a role in damage recognition or damage verification, may function as a processivity factor.     

On the Enzymatic Properties of Dnmt1: Specificity,Processivity, Mechanism of Linear Diffusion and Allosteric Regulation of the Enzyme

In this short review the enzymatic properties of Dnmt1 are summarized. Studies on the specificity of Dnmt1 have shown that the enzymes has 30-40 fold preference for hemimethylated target sites. It methylates hemimethylated DNA in a processive reaction, moving on the DNA in a random walk. Binding of DNA to allosteric site(s) in the N-terminal part of the enzyme can lead to stimulation and inhibition of its catalytic activity depending on the nature of the substrate and effector.     

Specificity of DNA binding and methylation by the M.FokI DNA methyltransferase

The M.FokI adenine-N(6) DNA methyltransferase recognizes the asymmetric DNA sequence GGATG/CATCC. It consists of two domains each containing all motifs characteristic for adenine-N(6) DNA methyltransferases. We have studied the specificity of DNA-methylation by both domains using 27 hemimethylated oligonucleotide substrates containing recognition sites which differ in one or two base pairs from GGATG or CATCC. The N-terminal domain of M.FokI interacts very specifically with GGATG-sequences, because only one of the altered sites is modified. In contrast, the C-terminal domain shows lower specificity. It prefers CATCC-sequences but only two of the 12 star sites (i.e. sites that differ in 1 bp from the recognition site) are not accepted and some star sites are modified with rates reduced only 2-3-fold. In addition, GGATGC- and CGATGC-sites are modified which differ at two positions from CATCC. DNA binding experiments show that the N-terminal domain preferentially binds to hemimethylated GGATG/C(m)ATCC sequences whereas the C-terminal domain binds to DNA with higher affinity but without specificity. Protein-protein interaction assays show that both domains of M.FokI are in contact with each other. However, several DNA-binding experiments demonstrate that DNA-binding of both domains is mutually exclusive in full-length M.FokI and both domains do not functionally influence each other. The implications of these results on the molecular evolution of type IIS restriction/modification systems are discussed.     

Processive proofreading is intrinsic to T4 DNA polymerase.

DNA replication occurs in vivo with very high processivity, meaning that the replication complex assembles at the origin(s) of replication and then performs template-directed synthesis of DNA over virtually the entire genome without dissociation. Such processivity also characterizes reconstituted replication holoenzyme complexes in vitro. However, the isolated DNA polymerases are much less processive, especially under physiological conditions. In this paper we monitor the degree of processivity displayed by the bacteriophage T4-coded DNA polymerase while in its proofreading mode by asking whether an isolated polymerase can "edit-out" the 3'-terminal nucleotide from the primer (using the 3'----5'-exonuclease activity of the polymerase) and then switch into the synthesis mode without dissociating from the DNA template. This "switch experiment" is accomplished by using mismatched primer/template substrates as an experimental tool to mimic the situation that T4 DNA polymerase encounters after a misincorporation event has occurred. By performing experiments under single-turnover conditions (obtained using a heparin trap), we demonstrate that T4 DNA polymerase, upon encountering a misincorporated base, neither synthesizes the next base nor dissociates into solution. Instead, with a greater than 80% probability, it removes the misincorporated base and then continues synthesis in a fully processive manner. We also show that the removal of a doubly mispaired sequence from the 3'-terminus of the primer, followed by synthesis, is comparably processive. In contrast, the apparent processivity of removing a triply mispaired terminus is much reduced. Taken together, these observations are consistent with the notion that the "editing active site" of the T4 enzyme optimally accommodates only two unpaired nucleotide residues. Our results do not support the idea that the exonuclease activity of T4 DNA polymerase is highly selective for mismatched termini; they suggest instead that the dwell time at a misincorporated base determines overall editing efficiency. The integrated results of this study provide additional insight into the structure of the T4 DNA polymerase, as well as into the interactions between the polymerase and the polymerase accessory proteins that are required to provide the holoenzyme complex with full processivity.     

Hallmarks of Processivity in Glycoside Hydrolases from Crystallographic and Computational Studies of the Serratia marcescens Chitinases

Degradation of recalcitrant polysaccharides in nature is typically accomplished by mixtures of processive and nonprocessive glycoside hydrolases (GHs), which exhibit synergistic activity wherein nonprocessive enzymes provide new sites for productive attachment of processive enzymes. GH processivity is typically attributed to active site geometry, but previous work has demonstrated that processivity can be tuned by point mutations or removal of single loops. To gain additional insights into the differences between processive and nonprocessive enzymes that give rise to their synergistic activities, this study reports the crystal structure of the catalytic domain of the GH family 18 nonprocessive endochitinase, ChiC, from Serratia marcescens. This completes the structural characterization of the co-evolved chitinolytic enzymes from this bacterium and enables structural analysis of their complementary functions. The ChiC catalytic module reveals a shallow substrate-binding cleft that lacks aromatic residues vital for processivity, a calcium-binding site not previously seen in GH18 chitinases, and, importantly, a displaced catalytic acid (Glu-141), suggesting flexibility in the catalytic center. Molecular dynamics simulations of two processive chitinases (ChiA and ChiB), the ChiC catalytic module, and an endochitinase from Lactococcus lactis show that the nonprocessive enzymes have more flexible catalytic machineries and that their bound ligands are more solvated and flexible. These three features, which relate to the more dynamic on-off ligand binding processes associated with nonprocessive action, correlate to experimentally measured differences in processivity of the S. marcescens chitinases. These newly defined hallmarks thus appear to be key dynamic metrics in determining processivity in GH enzymes complementing structural insights.     

Identification of the active oligomeric state of an essential adenine DNA methyltransferase from Caulobacter crescentus

Caulobacter crescentus contains one of the two known prokaryotic DNA methyltransferases that lacks a cognate endonuclease. This endogenous cell cycle regulated adenine DNA methyltransferase (CcrM) is essential for C. crescentus cellular viability. DNA methylation catalyzed by CcrM provides an obligatory signal for the proper progression through the cell cycle. To further our understanding of the regulatory role played by CcrM, we sought to investigate its biophysical properties. In this paper we employed equilibrium ultracentrifugation, velocity ultracentrifugation, and chemical cross-linking to show that CcrM is dimeric at physiological concentrations. However, surface plasmon resonance experiments in the presence of S-adenosyl-homocysteine evince that CcrM binds as a monomer to a defined hemi-methylated DNA substrate containing the canonical methylation sequence, GANTC. Initial velocity experiments demonstrate that dimerization of CcrM does not affect DNA methylation. Collectively, these findings suggest that CcrM is active as a monomer and provides a possible in vivo role for dimerization as a means to stabilize CcrM from premature catabolism.     

Single fungal kinesin motor molecules move processively along microtubules

Conventional kinesins are two-headed molecular motors that move as single molecules micrometer-long distances on microtubules by using energy derived from ATP hydrolysis. The presence of two heads is a prerequisite for this processive motility, but other interacting domains, like the neck and K-loop, influence the processivity and are implicated in allowing some single-headed kinesins to move processively. Neurospora kinesin (NKin) is a phylogenetically distant, dimeric kinesin from Neurospora crassa with high gliding speed and an unusual neck domain. We quantified the processivity of NKin and compared it to human kinesin, HKin, using gliding and fluorescence-based processivity assays. Our data show that NKin is a processive motor. Single NKin molecules translocated microtubules in gliding assays on average 2.14 micro m (N = 46). When we tracked single, fluorescently labeled NKin motors, they moved on average 1.75 micro m (N = 182) before detaching from the microtubule, whereas HKin motors moved shorter distances (0.83 micro m, N = 229) under identical conditions. NKin is therefore at least twice as processive as HKin. These studies, together with biochemical work, provide a basis for experiments to dissect the molecular mechanisms of processive movement.     

The DNA scanning mechanism of T4 endonuclease V. Effect of NaCl concentration on processive nicking activity

T4 endonuclease V is a pyrimidine dimer-specific endonuclease which generates incisions in DNA at the sites of pyrimidine dimers by a processive reaction mechanism. A model is presented in which the degree of processivity is directly related to the efficacy of the one-dimensional diffusion of endonuclease V on DNA by which the enzyme locates pyrimidine dimers. The modulation of the processive nicking activity of T4 endonuclease V on superhelical covalently closed circular DNA (form I) which contains pyrimidine dimers has been investigated as a function of the ionic strength of the reaction. Agarose gel electrophoresis was used to separate the three topological forms of the DNA which were generated in time course reactions of endonuclease V with dimer-containing form I DNA in the absence of NaCl, and in 25, 50, and 100 mM NaCl. The degree of processivity was evaluated in terms of the mass fraction of form III (linear) DNA which was produced as a function of the fraction of form I DNA remaining. Processivity is maximal in the absence of NaCl and decreases as the NaCl concentration is increased. At 100 mM NaCl, processivity is abolished and endonuclease V generates incisions in DNA at the site of dimers by a distributive reaction mechanism. The change from the distributive to a processive reaction mechanism occurs at NaCl concentrations slightly below 50 mM. The high degree of processivity which is observed in the absence of NaCl is reversible to the distributive mechanism, as demonstrated by experiments in which the NaCl concentration was increased during the time course reaction. In addition, unirradiated DNA inhibited the incision of irradiated DNA only at NaCl concentrations at which processivity was observed.     

Facilitated diffusion during catalysis by EcoRI endonuclease. Nonspecific interactions in EcoRI catalysis

The potential for processive EcoRI endonuclease hydrolysis has been examined on several DNA substrates containing two EcoRI sites which were embedded in identical sequence environments. With a 388-base pair circular DNA, in which the two recognition sites are separated by 51 base pairs (shorter distance) or 337 base pairs (longer distance), 77 and 34% of all events involved processive hydrolysis at ionic strengths of 0.059 and 0.13, respectively. However, the frequency of processive action on linear substrates, in which the two sites were separated by 51 base pairs, was only 42 and 17% at these ionic strengths, values half those observed with the circular DNA. Processive action was not detectable on circular or linear substrates at an ionic strength of 0.23. These findings indicate that DNA search by the endonuclease occurs by facilitated diffusion, a mechanism in which the protein locates and leaves its recognition sequence by interacting with nonspecific DNA sites. We suggest that processivity on linear substrates is limited to values half that for small circles due to partitioning of the enzyme between the two products generated by cleavage of a linear molecule. Given such topological effects, measured processivity values imply that the endonuclease can diffuse within a DNA domain to locate and recognize an EcoRI site 50 to 300 base pairs distant from an initial binding site, with minimum search efficiencies being 80 and 30% at ionic strengths of 0.059 and 0.13, respectively. The high efficiency of processive action indicates that a positionally correlated mode of search plays a major role in facilitated diffusion in this system under such conditions. Also consistent with this view was the identification of a striking position effect when two closely spaced EcoRI sites were asymmetrically positioned near the end of a linear DNA. The endonuclease displays a substantial preference for the more centrally located recognition sequence. This preference does not reflect differential sensitivity of the two sites to cleavage per se, but can be simply explained by preferential entry of the enzyme via the larger nonspecific target available to the more centrally positioned recognition sequence. These conclusions differ from those of a previous qualitative analysis of endonuclease processivity over short distances (Langowski, J., Alves, J., Pingoud, A., and Maass, G. (1983) Nucleic Acids Res. 11, 501-513).     

Evaluation of the role of the vaccinia virus uracil DNA glycosylase and A20 proteins as intrinsic components of the DNA polymerase holoenzyme

The vaccinia virus DNA polymerase is inherently distributive but acquires processivity by associating with a heterodimeric processivity factor comprised of the viral A20 and D4 proteins. D4 is also an enzymatically active uracil DNA glycosylase (UDG). The presence of an active repair protein as an essential component of the polymerase holoenzyme is a unique feature of the replication machinery. We have shown previously that the A20-UDG complex has a stoichiometry of ～1:1, and our data suggest that A20 serves as a bridge between polymerase and UDG. Here we show that conserved hydrophobic residues in the N' terminus of A20 are important for its binding to UDG. Our data argue against the assembly of D4 into higher order multimers, suggesting that the processivity factor does not form a toroidal ring around the DNA. Instead, we hypothesize that the intrinsic, processive DNA scanning activity of UDG tethers the holoenzyme to the DNA template. The inclusion of UDG as an essential holoenzyme component suggests that replication and base excision repair may be coupled. Here we show that the DNA polymerase can utilize dUTP as a substrate in vitro. Moreover, uracil moieties incorporated into the nascent strand during holoenzyme-mediated DNA synthesis can be excised by the viral UDG present within this holoenzyme, leaving abasic sites. Finally, we show that the polymerase stalls upon encountering an abasic site in the template strand, indicating that, like many replicative polymerases, the poxviral holoenzyme cannot perform translesion synthesis across an abasic site.     

Bacteriophage T4 Dam DNA-(N6-adenine)-methyltransferase. Processivity and orientation to the methylation target

We carried out steady state and pre-steady state (burst) kinetic analyses of the bacteriophage T4 Dam DNA-(N(6)-adenine)-methyltransferase (MTase)-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to Ade in oligonucleotide duplexes containing one or two specific GATC sites with different combinations of methylated and unmodified targets. We compared the results for ligated 40-mer duplexes with those of the mixtures of the two unligated duplexes used to generate the 40-mers. The salient results are as follows: (i) T4 Dam MTase modifies 40-mer duplexes in a processive fashion. (ii) During processive movement, T4 Dam rapidly exchanges product S-adenosyl-l-homocysteine (AdoHcy) for substrate AdoMet without dissociating from the DNA duplex. (iii) T4 Dam processivity is consistent with an ordered bi-bi mechanism AdoMet downward arrow DNA downward arrow DNA(Me) upward arrow AdoHcy upward arrow. However, in contrast to the steady state, here DNA(Me) upward arrow signifies departure from a methylated site GMTC upward arrow without physically dissociating from the DNA. (iv) Following methyl transfer at one site and linear diffusion to a hemimethylated site, a reconstituted T4 Dam-AdoMet complex rapidly reorients itself to the (productive) unmethylated strand. T4 Dam-AdoHcy cannot reorient at an enzymatically created GMTC site. (v) The inhibition potential of fully methylated sites 5'-GMTC/5'-GMTC is much lower for a long DNA molecule compared with short single-site duplexes.     

Tobacco proliferating cell nuclear antigen binds directly and stimulates both activity and processivity of ddNTP-sensitive mungbean DNA polymerase

PCNA is well known as a component of DNA replication system and plays important roles in multiple cellular pathways in addition to replication and repair. In this work we have demonstrated the physical and functional interaction between tobacco PCNA and mungbean ddNTP-sensitive DNA polymerase which shares many physicochemical properties with family X-DNA polymerases except with the moderately processive mode of nucleotide incorporation. We have shown here that recombinant PCNA binds directly to mungbean DNA polymerase as revealed in affinity chromatography, pull-down and co-immunoprecipitation approaches. In vitro DNA polymerase activity assay and processivity analyses indicated recombinant PCNA specifically stimulates both activity and processivity of mungbean DNA polymerase. These observations lead to interesting speculation about the functional significance of the ddNTP-sensitive enzyme in replication event in higher plants since the enzyme has been shown to be active and expressed at an elevated level during the endoreduplication stages in developing mungbean seeds.     

Cooperative DNA Binding and Protein/DNA Fiber Formation Increases the Activity of the Dnmt3a DNA Methyltransferase

The Dnmt3a DNA methyltransferase has been shown to bind cooperatively to DNA and to form large multimeric protein/DNA fibers. However, it has also been reported to methylate DNA in a processive manner, a property that is incompatible with protein/DNA fiber formation. We show here that the DNA methylation rate of Dnmt3a increases more than linearly with increasing enzyme concentration on a long DNA substrate, but not on a short 30-mer oligonucleotide substrate. We also show that addition of a catalytically inactive Dnmt3a mutant, which carries an amino acid exchange in the catalytic center, increases the DNA methylation rate by wild type Dnmt3a on the long substrate but not on the short one. In agreement with this finding, preincubation experiments indicate that stable protein/DNA fibers are formed on the long, but not on the short substrate. In addition, methylation experiments with substrates containing one or two CpG sites did not provide evidence for a processive mechanism over a wide range of enzyme concentrations. These data clearly indicate that Dnmt3a binds to DNA in a cooperative reaction and that the formation of stable protein/DNA fibers increases the DNA methylation rate. Fiber formation occurs at low μm concentrations of Dnmt3a, which are in the range of Dnmt3a concentrations in the nucleus of embryonic stem cells. Understanding the mechanism of Dnmt3a is of vital importance because Dnmt3a is a hotspot of somatic cancer mutations one of which has been implicated in changing Dnmt3a processivity.     

Effects of substitutions of arginine residues on the basic surface of herpes simplex virus UL42 support a role for DNA binding in processive DNA synthesis

The way that UL42, the processivity subunit of the herpes simplex virus DNA polymerase, interacts with DNA and promotes processivity remains unclear. A positively charged face of UL42 has been proposed to participate in electrostatic interactions with DNA that would tether the polymerase to a template without preventing its translocation via DNA sliding. An alternative model proposes that DNA binding by UL42 is not important for processivity. To investigate these issues, we substituted alanine for each of four conserved arginine residues on the positively charged surface. Each single substitution decreased the DNA binding affinity of UL42, with 14- to 30-fold increases in apparent dissociation constants. The mutant proteins exhibited no meaningful change in affinity for binding to the C terminus of the catalytic subunit of the polymerase, indicating that the substitutions exert a specific effect on DNA binding. The substitutions decreased UL42-mediated long-chain DNA synthesis by the polymerase in the same rank order in which they affected DNA binding, consistent with a role for DNA binding in polymerase processivity. Combining these substitutions decreased DNA binding further and impaired the complementation of a UL42 null virus in transfected cells. Additionally, using a revised mathematical model to analyze rates of dissociation of UL42 from DNAs of various lengths, we found that dissociation from internal sites, which would be the most important for tethering the polymerase, was relatively slow, even at ionic strengths that permit processive DNA synthesis by the holoenzyme. These data provide evidence that the basic surface of UL42 interacts with DNA and support a model in which DNA binding by UL42 is important for processive DNA synthesis.     

A protein that binds to the P1 origin core and the oriC 13mer region in a methylation-specific fashion is the product of the host seqA gene.

The P1 plasmid replication origin P1oriR is controlled by methylation of four GATC adenine methylation sites within heptamer repeats. A comparable (13mer) region is present in the host origin, oriC. The two origins show comparable responses to methylation; negative control by recognition of hemimethylated DNA (sequestration) and a positive requirement for methylation for efficient function. We have isolated a host protein that recognizes the P1 origin region only when it is isolated from a strain proficient for adenine methylation. The substantially purified 22 kDa protein also binds to the 13mer region of oriC in a methylation-specific fashion. It proved to be the product of the seqA gene that acts in the negative control of oriC by sequestration. We conclude that the role of the SeqA protein in sequestration is to recognize the methylation state of P1oriR and oriC by direct DNA binding. Using synthetic substrates we show that SeqA binds exclusively to the hemimethylated forms of these origins forms that are the immediate products of replication in a methylation-proficient strain. We also show that the protein can recognize sequences with multiple GATC sites, irrespective of the surrounding sequence. The basis for origin specificity is primarily the persistence of hemimethylated forms that are over-represented in the natural. DNA preparations relative to controls.