DNA-methyltransferase SsoII as a bifunctional protein: Features of the interaction with the promoter region of SsoII restriction-modification genes

DNA duplexes bearing an aldehyde group at the 2'-position of the sugar moiety were used for affinity modification of (cytosine-5)-DNA methyltransferase SsoII. It is shown that lysine residues of M.SsoII N-terminal region are located in proximity to DNA sugar-phosphate backbone of a regulatory sequence of promoter region of SsoII restriction-modification enzyme coding genes. The ability of the two M.SsoII subunits to interact with DNA regulatory sequence has been demonstrated by affinity modification using DNA duplexes with two 2'-aldehyde groups. Changes in nucleotide sequence of one half of the regulatory region prevented cross-linking of the second M.SsoII subunit. The results on sequential affinity modification of M.SsoII by two types of modified DNA ligands (i.e. by 2'-aldehyde-containing and phosphoryldisulfide-containing) have demonstrated the possibility of covalent attachment of the protein to two different DNA recognition sites: regulatory sequence and methylation site.     

Tumor promotion resistant cells are deficient in AP-1 DNA binding, JunD DNA binding and JunD expression and form different AP-1-DNA complexes than promotion sensitive cells

The JB6 cell culture model is used to identify molecular determinants of susceptibility to the promotion of neoplastic transformation. Clonal variants susceptible to transformation ('P+' cells) form numerous anchorage-independent colonies in soft agar upon treatment with the phorbol ester tumor promoter TPA, whereas resistant variants ('P-' cells) do not. We now report that there is significantly less binding of activator protein-1 (AP-1) to its DNA binding site in P- cells than in P+ cells. Gel supershift assays were performed to detect association of all seven AP-1 family members with their DNA binding site in TPA-treated and -untreated P+ and P- cells. Significantly lower DNA binding and protein expression of JunD were detected in P- cells than in P+ cells. c-Jun was detected in P+, but not P-, AP-1-DNA complexes, and c-Fos was detected in P-, but not P+, AP-1-DNA complexes. These and other phenotype-specific differences in abundance and composition of AP-1-DNA complexes may play a role in the resistance of P- cells to tumor promoter-induced transformation.     

Second monomer binding is the rate-limiting step in the formation of the dimeric PhoP-DNA complex

PhoP, the response regulator of the PhoP/PhoQ system, regulates Mg(2+) homeostasis in Salmonella typhimurium. Dimerization of PhoP on the DNA is necessary for its regulatory function, and PhoP regulates the expression of genes in a phosphorylation-dependent manner. Higher PhoP concentrations, however, can activate PhoP and substitute for phosphorylation-dependent gene regulation. Activation of PhoP by phosphorylation is explained by self-assembly of phosphorylated PhoP (PhoP-p) in solution and binding of the PhoP-p dimer to the promoter. To understand the mechanism of PhoP dimerization on the DNA, we examined the interactions of PhoP with double-stranded DNAs containing the canonical PhoP box (PB). We present results from multiple biophysical methods, demonstrating that PhoP is a monomer in solution over a range of concentrations and binds to PB in a stepwise manner with a second PhoP molecule binding weakly. The affinity for the binding of the first PhoP molecule to PB is more than ～17-fold higher than the affinity of the second PhoP monomer for PB. Kinetic analyses of PhoP binding reveal that the on rate of the second PhoP monomer binding is the rate-limiting step during the formation of the (PhoP)(2)-DNA complex. Results show that a moderate increase in PhoP concentration can promote dimerization of PhoP on the DNA, which otherwise could be achieved by PhoP-p at much lower protein concentrations. Detailed analyses of PhoP-DNA interactions have revealed the existence of a kinetic barrier that is the key for specificity in the formation of the productive (PhoP)(2)-DNA complex.     

Reaction of the antitumor antibiotic CC-1065 with DNA. Location of the site of thermally induced strand breakage and analysis of DNA sequence specificity

CC-1065 is a unique antitumor antibiotic produced by Streptomyces zelensis. The potent cytotoxic effects of this drug are thought to be due to its ability to form a covalent adduct with DNA through N3 of adenine. Thermal treatment of CC-1065-DNA adducts leads to DNA strand breakage. We have shown that the CC-1065 structural modification of DNA that leads to DNA strand breakage is related to the primary alkylation site on DNA. The thermally induced DNA strand breakage occurs between the deoxyribose at the adenine covalent binding site and the phosphate on the 3' side. No residual modification of DNA is detected on the opposite strand around the CC-1065 lesion. Using the early promoter element of SV40 DNA as a target, we have examined the DNA sequence specificity of CC-1065. A consensus sequence analysis of CC-1065 binding sites on DNA reveals two distinct classes of sequences for which CC-1065 is highly specific, i.e., 5'PuNTTA and 5'AAAAA. The orientation of the DNA sequence specificity relative to the covalent binding site provides a basis for predicting the polarity of drug binding in the minor groove. Stereo drawings of the CC-1065-DNA adduct are proposed that are predictive of features of the CC-1065-DNA adduct elucidated in this investigation.     

G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA

It has been previously shown that the DHX36 gene product, G4R1/RHAU, tightly binds tetramolecular G4-DNA with high affinity and resolves these structures into single strands. Here, we test the ability of G4R1/RHAU to bind and unwind unimolecular G4-DNA. Gel mobility shift assays were used to measure the binding affinity of G4R1/RHAU for unimolecular G4-DNA-formed sequences from the Zic1 gene and the c-Myc promoter. Extremely tight binding produced apparent K_d’s of 6, 3 and 4 pM for two Zic1 G4-DNAs and a c-Myc G4-DNA, respectively. The low enzyme concentrations required for measuring these K_d’s limit the precision of their determination to upper boundary estimates. Similar tight binding was not observed in control non-G4 forming DNA sequences or in single-stranded DNA having guanine-rich runs capable of forming tetramolecular G4-DNA. Using a peptide nucleic acid (PNA) trap assay, we show that G4R1/RHAU catalyzes unwinding of unimolecular Zic1 G4-DNA into an unstructured state capable of hybridizing to a complementary PNA. Binding was independent of adenosine triphosphate (ATP), but the PNA trap assay showed that unwinding of G4-DNA was ATP dependent. Competition studies indicated that unimolecular Zic1 and c-Myc G4-DNA structures inhibit G4R1/RHAU-catalyzed resolution of tetramolecular G4-DNA. This report provides evidence that G4R1/RHAU tightly binds and unwinds unimolecular G4-DNA structures.     

The new RENT family of repetitive elements in Nicotiana species harbors gene regulatory elements related to the tCUP cryptic promoter.

The tCUP cryptic constitutive promoter was discovered in the tobacco genome by T-DNA (transfer DNA) tagging with a promoterless GUS-nos gene. Here, we show that the portion of the tCUP sequence containing a variety of cryptic gene regulatory elements is related to a new family of moderately repetitive sequences (10(2) copies), the RENT (repetitive element from Nicotiana tabacum) family. The RENT family is found only in certain Nicotiana species. Five RENT elements were cloned and sequenced. The RENT elements are a minimum of 5 kb in length and share 80-90% sequence similarity throughout their length. The 5' termini are the same in the isolated RENT family members and are characterized by a conserved border sequence (TGTTGA(T or C)ACCCAATTTT(T or C)). The 3' ends of RENT sequence similarity vary in location and sequence. The tCUP cryptic promoter originated from a unique truncated RENT element that interrupts a phytochelatin synthase-like gene that may have undergone rearrangements prior to or resulting from T-DNA insertion. No evidence was found for expressed coding regions within the RENT elements; however, like the cryptic gene regulatory elements within the tCUP sequence, the isolated RENT elements possess promoter activity and translational enhancer activity.     

Inhibition of murine DNA methyltransferase Dnmt3a by DNA duplexes containing pyrimidine-2(1<Emphasis Type="Italic">H</Emphasis>)-one

Here we studied the inhibition of the catalytic domain of Dnmt3a methyltransferase (Dnmt3a-CD) by DNA duplexes containing the mechanism-based inhibitor pyrimidine-2(1H)-one (P) instead of the target cytosine. It has been shown that conjugates of Dnmt3a-CD with P-DNA (DNA containing pyrimidine-2(1H)-one) are not stable to heating at 65°C in 0.1% SDS. The yield of covalent intermediate increases in the presence of the regulatory factor Dnmt3L. The importance of the DNA minor groove for covalent intermediate formation during the methylation reaction catalyzed by Dnmt3a-CD has been revealed. P-DNA was shown to inhibit Dnmt3a-CD; the IC₅₀ is 830 nM. The competitive mechanism of inhibition of Dnmt3a-CD by P-DNA has been elucidated. It is suggested that therapeutic effect of zebularine could be achieved by inhibition of not only Dnmt1 but also Dnmt3a.     

Stable association of viral protein VP1 with simian virus 40 DNA.

Mild dissociation of simian virus 40 particles releases a 110S virion core nucleoprotein complex containing histones and the three viral proteins VP1, VP2, and VP3. The association of viral protein VP1 within this nucleoprotein complex is mediated at least partially through a strong interaction with the viral DNA. Treatment of the virion-derived 110S nucleoprotein complex with 0.25% Sarkosyl dissociated VP2, VP3, and histones, leaving a stable VP1-DNA complex. The VP1-DNA complex had a sedimentation value of 30S and a density of 1.460 g/cm3. The calculated molecular weight of the complex was 7.9 x 10(6), with an average of 100 VP1 molecules per DNA. Agarose gel electrophoresis of the VP1-DNA complex demonstrated that VP1 is associated not only with form I and form II simian virus 40 DNAs but also with form III simian virus 40 DNA generated by cleavage with EcoRI.