Molecular Interactions of a DNA Modifying Enzyme APOBEC3F Catalytic Domain with a Single-Stranded DNA

The single-stranded DNA (ssDNA) cytidine deaminase APOBEC3F (A3F) deaminates cytosine (C) to uracil (U) and is a known restriction factor of HIV-1. Its C-terminal catalytic domain (CD2) alone is capable of binding single-stranded nucleic acids and is important for deamination. However, little is known about how the CD2 interacts with ssDNA. Here we report a crystal structure of A3F-CD2 in complex with a 10-nucleotide ssDNA composed of poly-thymine, which reveals a novel positively charged nucleic acid binding site distal to the active center that plays a key role in substrate DNA binding and catalytic activity. Lysine and tyrosine residues within this binding site interact with the ssDNA, and mutating these residues dramatically impairs both ssDNA binding and catalytic activity. This binding site is not conserved in APOBEC3G (A3G), which may explain differences in ssDNA-binding characteristics between A3F-CD2 and A3G-CD2. In addition, we observed an alternative Zn-coordination conformation around the active center. These findings reveal the structural relationships between nucleic acid interactions and catalytic activity of A3F.     

Ff gene 5 protein has a high binding affinity for single-stranded phosphorothioate DNA

The gene 5 protein (g5p) of Ff bacteriophages is a well-studied model ssDNA-binding protein that binds cooperatively to the Ff ssDNA genome and single-stranded polynucleotides. Its affinity, K omega (the intrinsic binding constant times a cooperativity factor), can differ by several orders of magnitude for ssDNAs of different nearest-neighbor base compositions [Mou, T. C., Gray, C. W., and Gray, D. M. (1999) Biophys. J. 76, 1537-1551]. We found that the DNA backbone can also dramatically affect the binding affinity. The K omega for binding phosphorothioate-modified S-d(A)(36) was >300-fold higher than for binding unmodified P-d(A)(36) at 0.2 M NaCl. CD titrations showed that g5p bound phosphorothioate-modified oligomers with the same stoichiometry as unmodified oligomers. The CD spectrum of S-d(A)(36) underwent the same qualitative change upon protein binding as did the spectrum of unmodified DNA, and the phosphorothioate-modified DNA appeared to bind in the normal g5p binding site. Oligomers of d(A)(36) with different proportions of phosphorothioate nucleotides had binding affinities and CD perturbations intermediate to those of the fully modified and unmodified sequences. The influence of phosphorothioation on binding affinity was nearly proportional to the extent of the modification, with a small nearest-neighbor dependence. These and other results using d(ACC)(12) oligomers and mutant proteins indicated that the increased binding affinity of g5p for phosphorothioate DNA was not a polyelectrolyte effect and probably was not an effect due to the altered nucleic acid structure, but was more likely a general effect of the properties of the sulfur in the context of the phosphorothioate group.     

Mechanism of allosteric regulation of Dnmt1's processivity

We have analyzed the relationship between the allosteric regulation and processive catalysis of DNA methyltransferase 1 (Dnmt1). Processivity is described quantitatively in terms of turnover rate, DNA dissociation rate, and processivity probability. Our results provide further evidence that the active site and the allosteric sites on Dnmt1 can bind DNA independently. Dnmt1's processive catalysis on unmethylated DNA is partially inhibited when the allosteric site binds unmethylated DNA and fully inhibited when the allosteric site binds a single-stranded oligonucleotide inhibitor. The partial inhibition by unmethylated DNA is caused by a decrease in the turnover rate and an increase in the substrate DNA dissociation rate. Processive catalysis with premethylated DNA is not affected if the allosteric site is exposed to premethylated DNA but is fully inhibited if the allosteric site binds unmethylated DNA or poly(dA-dT). In sum, the occupancy of the allosteric site modulates the enzyme's commitment to catalysis, which reflects the nature of the substrate and the DNA bound at the allosteric site. Our in vitro results are consistent with the possibility that the processive action of Dnmt1 may be regulated in vivo by specific regulatory nucleic acids such as DNA, RNA, or poly(ADP-ribose).     

Red light-activated phosphorothioate oligodeoxyribonucleotides

Hairpin-structured phosphorothioate oligodeoxyribonucleotides containing a singlet oxygen-sensitive linker in the loop were prepared. These compounds do not bind complementary nucleic acids in the dark. Upon irradiation with red light in the presence of chlorine e6 the linker within these compounds is cleaved and a single-stranded oligodeoxyribonucleotide is produced. The latter compound is an efficient binder of complementary nucleic acids. This is the first example of ‘caged’ phosphorothioate oligodeoxyribonucleotides, whose nucleic acid binding ability is triggered by red light.     

Topography of the interaction of recA protein with single-stranded deoxyoligonucleotides

recA protein, in the presence of adenosine 5'-(gamma-thio)triphosphate, formed stable complexes with single-stranded deoxyoligonucleotides between 9 and 20 residues in length but not with those 8-residues long. The binding of recA protein to a 15-mer and 20-mer completely protected the sugar-phosphate backbone of the nucleic acid from digestion by pancreatic deoxyribonuclease I and protected the 5'-terminal phosphate from cleavage by calf intestinal alkaline phosphatase. Ethylation of the phosphate backbone at any position by ethylnitrosourea blocked the binding of recA protein to the 15-mer but not to the 20-mer. Ethylation of phosphates near the ends of the 15-mer interfered less, suggesting a minimum binding site requirement. In contrast to the protection of the nucleic acid backbone, recA protein did not protect the N-7 position of guanine or the N-3 position of adenine from methylation by dimethyl sulfate, but rather enhanced the methylation of guanine. These results indicate that recA protein binds primarily to the phosphate backbone of single-stranded DNA, leaving the bases free for homologous pairing. We present a model for the organization of the presynaptic filament.     

The Nucleic Acid Binding Activity of Bleomycin Hydrolase Is Involved in Bleomycin Detoxification

Yeast bleomycin hydrolase, Gal6p, is a cysteine peptidase that detoxifies the anticancer drug bleomycin. Gal6p is a dual-function protein capable of both nucleic acid binding and peptide cleavage. We now demonstrate that Gal6p exhibits sequence-independent, high-affinity binding to single-stranded DNA, nicked double-stranded DNA, and RNA. A region of the protein that is involved in binding both RNA and DNA substrates is delineated. Immunolocalization reveals that the Gal6 protein is chiefly cytoplasmic and thus may be involved in binding cellular RNAs. Variant Gal6 proteins that fail to bind nucleic acid also exhibit reduced ability to protect cells from bleomycin toxicity, suggesting that the nucleic acid binding activity of Gal6p is important in bleomycin detoxification and may be involved in its normal biological functions.     

Stoichiometry and specificity of binding of Rauscher oncovirus 10,000-dalton (p10) structural protein to nucleic acids.

A structural protein of Rauscher oncovirus of about 8,000 to 10,000 daltons (p10), encoded by the gag gene, has been purified in high yield to apparent homogeneity by a simple three-step procedure. The purified protein was highly basic, with an isoelectric point of more than 9.0, and its immunological antigenicity was chiefly group specific. A distinctive property of the protein was the binding to nucleic acids. The stoichiometry of p10 binding to Rauscher virus RNA was analyzed using both 125I-labeled p10 and 3H-labeled RNA. The protein-RNA complex, cross-linked by formaldehyde, was separated from free RNA and free protein by velocity sedimentation and density gradient centrifugation. A maximum of about 140 mol of p10 was bound per mol of 35S RNA, or about one molecule of p10 per 70 nucleotides. This protein-RNA complex banded at a density of about 1.55 g/ml. The number of nucleic acid sites bound and the affinity of p10 binding differed significantly among the other polynucleotides tested. The protein bound to both RNA and DNA with a preference for single-stranded molecules. Rauscher virus RNA and single-stranded phage fd DNA contained the highest number of binding sites. Binding to fd DNA was saturated with about 30 mol of p10 per mol of fd DNA, an average of about one p10 molecule per 180 nucleotides. The apparent binding constant was 7.3 X 10(7) M(-1). The properties of the p10 place it in a category with other nucleic acid binding proteins that achieve a greater binding density on single-stranded than on double-stranded molecules and appear to act by facilitating changes in polynucleotide conformation.     

Theory of electrostatically regulated binding of T4 gene 32 protein to single- and double-stranded DNA

Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA binding protein, which is essential for DNA replication, recombination, and repair. In a recent article, we described a new method using single DNA molecule stretching measurements to determine the noncooperative association constants K(ds) to double-stranded DNA for gp32 and *I, a truncated form of gp32. In addition, we developed a single molecule method for measuring K(ss), the association constant of these proteins to single-stranded DNA. We found that in low salt both K(ds) and K(ss) have a very weak salt dependence for gp32, whereas for *I the salt dependence remains strong. In this article we propose a model that explains the salt dependence of gp32 and *I binding to single-stranded nucleic acids. The main feature of this model is the strongly salt-dependent removal of the C-terminal domain of gp32 from its nucleic acid binding site that is in pre-equilibrium to protein binding to both double-stranded and single-stranded nucleic acid. We hypothesize that unbinding of the C-terminal domain is associated with counterion condensation of sodium ions onto this part of gp32, which compensates for sodium ion release from the nucleic acid upon its binding to the protein. This results in the salt-independence of gp32 binding to DNA in low salt. The predictions of our model quantitatively describe the large body of thermodynamic and kinetic data from bulk and single molecule experiments on gp32 and *I binding to single-stranded nucleic acids.     

DNA cytosine C5 methyltransferase Dnmt1: catalysis-dependent release of allosteric inhibition

We followed the cytosine C(5) exchange reaction with Dnmt1 to characterize its preference for different DNA substrates, its allosteric regulation, and to provide a basis for comparison with the bacterial enzymes. We determined that the methyl transfer is rate-limiting, and steps up to and including the cysteine-cytosine covalent intermediate are in rapid equilibrium. Changes in these rapid equilibrium steps account for many of the previously described features of Dnmt1 catalysis and specificity including faster reactions with premethylated DNA versus unmethylated DNA, faster reactions with DNA in which guanine is replaced with inosine [poly(dC-dG) vs poly(dI-dC)], and 10-100-fold slower catalytic rates with Dnmt1 relative to the bacterial enzyme M.HhaI. Dnmt1 interactions with the guanine within the CpG recognition site can prevent the premature release of the target base and solvent access to the active site that could lead to mutagenic deamination. Our results suggest that the beta-elimination step following methyl transfer is not mediated by free solvent. Dnmt1 shows a kinetic lag in product formation and allosteric inhibition with unmethylated DNA that is not observed with premethylated DNA. Thus, we suggest the enzyme undergoes a slow relief from allosteric inhibition upon initiation of catalysis on unmethylated DNA. Notably, this relief from allosteric inhibition is not caused by self-activation through the initial methylation reaction, as the same effect is observed during the cytosine C(5) exchange reaction in the absence of AdoMet. We describe limitations in the Michaelis-Menten kinetic analysis of Dnmt1 and suggest alternative approaches.     

N-hydroxy-4-aminobiphenyl-DNA binding in human p53 gene: sequence preference and the effect of C5 cytosine methylation

4-Aminobiphenyl (4-ABP) is a major etiological agent for human bladder cancer. Metabolically activated 4-ABP is able to interact with DNA to form adducts that may induce mutations and initiate carcinogenesis. Thirty to sixty percent of bladder cancer has a mutation in the tumor suppressor p53 gene, and the mutational spectrum bears unique features. To date the DNA binding spectrum of 4-ABP in the p53 gene is not known due to the lack of methodology to detect 4-ABP-DNA adducts at nucleotide sequence level. We have found that UvrABC nuclease, a nucleotide excision repair complex isolated from Escherichia coli, is able to incise specifically and quantitatively DNA fragments modified with N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), an activated intermediate of 4-ABP. Using the UvrABC nuclease incision method, we mapped the binding spectrum of N-OH-4-ABP in DNA fragments containing exons 5, 7, and 8 of the human p53 gene and also determined the effect of C5 cytosine methylation on N-OH-4-ABP-DNA binding. We found that codon 285, a mutational hotspot at a non-CpG site in bladder cancer, is the preferential binding site for N-OH-4-ABP. We also found that C5 cytosine methylation greatly enhanced N-OH-4-ABP binding at CpG sites, and that two mutational hotspots at CpG sites, codons 175 and 248, became preferential binding sites for N-OH-4-ABP only after being methylated. These results suggest that both the unique DNA binding specificity of 4-ABP and cytosine methylation contribute to the mutational spectrum of the p53 gene in human bladder cancer.     

Identification of a second DNA binding site in human DNA methyltransferase 3A by substrate inhibition and domain deletion

The human DNA methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with μM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.     

Structural and enzymatic studies of the T4 DNA replication system. II. ATPase properties of the polymerase accessory protein complex

In this paper we report a detailed enzymatic characterization of the interaction of the polymerase accessory protein complex of the T4 DNA replication system with the various nucleic acid cofactors that activate the ATPase of the complex. We show that the ATPase activity of the T4 coded gene 44/62 protein complex is stimulated synergistically by binding of DNA and T4 gene 45 protein and that the level of ATPase activation appears to be directly correlated with the binding of nucleic acid cofactor. Binding of any partially or completely single-stranded DNA to the complete accessory protein complex increases the catalytic activity (as measured by Vmax) while decreasing the binding affinity for the ATP substrate. While single-stranded DNA is a moderately effective cofactor, we find that the optimal nucleic acid-binding site for the complex is the primer-template junction, rather than single-stranded DNA ends as previously reported in the literature. Gene 45 protein plays an essential role in directing the specificity of binding to primer-template sites, lowering the Km for primer-template sites almost 1000-fold, and increasing Vmax 100-fold, compared with the analogous values for gene 44/62 protein alone. The most effective primer-template site for binding and enzymatic activation has the physiologically relevant recessed 3'-OH configuration and an optimal size in excess of 18 base pairs of duplex DNA. We find that the chemical nature of the primer terminus (i.e. 3'-OH or 3'-H) does not affect the extent of ATPase activation and that binding of the polymerase accessory protein complex to DNA cofactors is salt concentration dependent but appreciably less so when the activating DNA is a primer-template junction. Finally, we show that the gene 32 protein (T4 coded single-stranded DNA-binding protein) can compete with the polymerase accessory protein complex for single-stranded DNA but not for the primer-template junction activation sites. The implications of these results for the structure and function of the polymerase accessory protein complex within the T4 DNA replication system are discussed.     

ATP binding modulates the nucleic acid affinity of hepatitis C virus helicase

The helicase of hepatitis C virus (HCV) unwinds nucleic acid using the energy of ATP hydrolysis. The ATPase cycle is believed to induce protein conformational changes to drive helicase translocation along the length of the nucleic acid. We have investigated the energetics of nucleic acid binding by HCV helicase to understand how the nucleotide ligation state of the helicase dictates the conformation of its nucleic acid binding site. Because most of the nucleotide ligation states of the helicase are transient due to rapid ATP hydrolysis, several compounds were analyzed to find an efficient unhydrolyzable ATP analog. We found that the beta-gamma methylene/amine analogs of ATP, ATPgammaS, or [AlF4]ADP were not effective in inhibiting the ATPase activity of HCV helicase. On the other hand, [BeF3]ADP was found to be a potent inhibitor of the ATPase activity, and it binds tightly to HCV helicase with a 1:1 stoichiometry. Equilibrium binding studies showed that HCV helicase binds single-stranded nucleic acid with a high affinity in the absence of ATP or in the presence of ADP. Upon binding to the ATP analog, a 100-fold reduction in affinity for ssDNA was observed. The reduction in affinity was also observed in duplex DNA with 3' single-stranded tail and in RNA but not in duplex DNA. The results of this study indicate that the nucleic acid binding site of HCV helicase is allosterically modulated by the ATPase reaction. The binding energy of ATP is used to bring HCV helicase out of a tightly bound state to facilitate translocation, whereas ATP hydrolysis and product release steps promote tight rebinding of the helicase to the nucleic acid. On the basis of these results we propose a Brownian motor model for unidirectional translocation of HCV helicase along the nucleic acid length.     

Hypomethylation of DNA during differentiation of friend erythroleukemia cells

DNA from mammalian cells has been shown to contain significant amounts of 5-methyl cytosine resulting from enzymatic transfer of methyl groups from s-adenosylmethionine to cytosine residues in the DNA polymer. The function of this modification is not known. We have found that DNA synthesized during chemically induced differentiation of friend erythroleukemia cells is hypomethylated, as measured by its ability to accept methyl groups transferred by homologous DNA methyltransferases in vitro. The extent of hypomethylation detected by this sensitive method is small, a decrease of less than 1.6 percent in 5-methylcytosine content. Hypomethylated DNA can be isolated from friend erythroleukemia cells grown in the presence of dimethyl sulfoxide, butyrate, hexamethylene-bis- acetamide, pentamethylene-bis acetamide, and ethionine. However, hypomethylated DNA is found only under conditions where differentiation is actually induced. DNA isolated from cells of a dimethyl sulfoxide- resistant subclone grown in the presence of that agent is not hypomethylated, although DNA of these cells becomes hypomethylated after growth in the presence of inducers that can trigger their differentiation. We also find that the DNA of friend erythroleukemia cells does not become hypomethylated when the cells are exposed to inducing agents in the presence of substances that inhibit differentiation. These results suggest a close link between genome modification by methylation and differentiation of friend erythroleukemia cells.     

Interaction of nucleolar phosphoprotein B23 with nucleic acids

The interaction of eukaryotic nucleolar phosphoprotein B23 with nucleic acids was examined by gel retardation and filter binding assays, by fluorescence techniques, and by circular dichroism. All studies utilized protein prepared under native conditions by a newly developed purification procedure. Electrophoretic gel mobility shift assays with phage M13 DNA suggested that protein B23 is a single-stranded nucleic acid binding protein. This was confirmed in competition binding assays with native or heat-denatured linearized plasmid pUC18 DNA where the protein showed a marked preference for the denatured form. In other competition assays, there was no apparent preference for single-stranded synthetic ribo- versus deoxyribonucleotides. Equilibrium binding with poly(riboethenoadenylic acid) indicated cooperative ligand binding with a protein binding site size of 11 nucleotides and an apparent binding constant (K omega) of 5 x 10(7) M-1 which includes an intrinsic binding constant (K) of 6.3 x 10(4) M-1 and a cooperativity factor (omega) of 800. In circular dichroism (CD) studies, protein B23, when combined with the single-stranded synthetic nucleic acids poly(rA) and poly(rC), effected a decrease in ellipticity and a shift of the positive peak at 260-270 nm toward higher wavelengths, indicating helix destabilizing activity. No CD changes were seen with double-stranded poly(dA.dT). The change in ellipticity of poly(rA) was sigmoidal upon addition of protein, confirming the cooperative behavior seen with fluorescence methods. These studies indicate that protein B23 binds cooperatively with high affinity for single-stranded nucleic acids and exhibits RNA helix destabilizing activity. These features may be related to its role in ribosome assembly.     

5'-Cytosine-phosphoguanine (CpG) methylation impacts the activity of natural and engineered meganucleases

In this study, we asked whether CpG methylation could influence the DNA binding affinity and activity of meganucleases used for genome engineering applications. A combination of biochemical and structural approaches enabled us to demonstrate that CpG methylation decreases I-CreI DNA binding affinity and inhibits its endonuclease activity in vitro. This inhibition depends on the position of the methylated cytosine within the DNA target and was almost total when it is located inside the central tetrabase. Crystal structures of I-CreI bound to methylated cognate target DNA suggested a molecular basis for such inhibition, although the precise mechanism still has to be specified. Finally, we demonstrated that the efficacy of engineered meganucleases can be diminished by CpG methylation of the targeted endogenous site, and we proposed a rational design of the meganuclease DNA binding domain to alleviate such an effect. We conclude that although activity and sequence specificity of engineered meganucleases are crucial parameters, target DNA epigenetic modifications need to be considered for successful gene editions.