A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.

The modulation of DNA-protein interactions by methylation of protein-binding sites in DNA and the occurrence in genomic imprinting, X chromosome inactivation, and fragile X syndrome of different methylation patterns in DNA of different chromosomal origin have underlined the need to establish methylation patterns in individual strands of particular genomic sequences. We report a genomic sequencing method that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA. The method utilizes bisulfite-induced modification of genomic DNA, under conditions whereby cytosine is converted to uracil, but 5-methylcytosine remains nonreactive. The sequence under investigation is then amplified by PCR with two sets of strand-specific primers to yield a pair of fragments, one from each strand, in which all uracil and thymine residues have been amplified as thymine and only 5-methylcytosine residues have been amplified as cytosine. The PCR products can be sequenced directly to provide a strand-specific average sequence for the population of molecules or can be cloned and sequenced to provide methylation maps of single DNA molecules. We tested the method by defining the methylation status within single DNA strands of two closely spaced CpG dinucleotides in the promoter of the human kininogen gene. During the analysis, we encountered in sperm DNA an unusual methylation pattern, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.     

Interaction of calicheamicin gamma1(I) and its related carbohydrates with DNA-protein complexes.

We report studies of the contribution of DNA structure, holding the sequence constant, to the affinity of calicheamicin gamma(1)(I) and its aryltetrasaccharide moiety for DNA. We used polynucleotide chains as models of known protein-binding sequences [the catabolite activator protein (CAP) consensus sequence, AP-1 and cAMP response element (CRE) sites] in their free and protein-bound forms. The proteins were selected to provide examples in which the minor-groove binding site for the carbohydrate is (CAP) or is not (GCN4) covered by the protein. Additionally, peptides related to the GCN4 and CREB families, which have different bending effects on their DNA-binding sites, were used. We observe that proteins of the CREB class, which induce a tendency to bend toward the minor groove at the center of the site, inhibit drug-cleavage sites located at the center of the free AP-1 or CRE DNA sites. In the case of GCN4, which does not induce DNA bending, there is no effect on calicheamicin cleavage of the CRE site, but we observe a GCN4-induced rearrangement of the cutting pattern in the AP-1 site. This effect may arise from either a subtle local conformational rearrangement not accompanied by bending or a localized reduction in DNA flexibility. Whereas GCN4 binding is not inhibited by the calicheamicin aryltetrasaccharide, binding of CAP to its DNA target is significantly inhibited, and calicheamicin cutting of DNA at the center of the CAP-DNA complex site is strongly reduced by protein binding. This result probably reflects steric inhibition of drug binding by the protein.     

Binding of the herpes simplex virus major regulatory protein to viral DNA.

Infected-cell protein 4 (ICP4), the major regulatory protein specified by herpes simplex virus 1 in infected cells, binds to homologs of the sequence ATCGTCnnnnYCGRC (A sites, where n is any nucleotide, Y is a pyrimidine, and R is a purine) and to unrelated sequences for which no consensus sequence has been derived (B sites). We have examined the binding of ICP4 to each of two A and two B binding sites by using Fab fragments of a monoclonal antibody that is reactive with an epitope located at the N terminus of ICP4 and that decreases the mobility of ICP4-DNA complexes in non-denaturing gels. The results indicate that each type of site binds two monomers of ICP4. Methylation-interference studies on the type B sites mapped the guanines whose methylation interfered with the binding of ICP4. The methylation-interference pattern obtained with one of the B sites was similar to that obtained on an A site but differed from that of the other B site. The ability of ICP4 to bind to DNA fragments containing the binding site appears to be dependent on length and on the proximity of the binding site to the fragment end. Short DNA fragments did not form stable complexes with ICP4 even though they contained all of the purines whose methylation interfered with the binding of the regulatory protein.     

Evidence for specific DNA sequences in the nuclear acceptor sites of the avian oviduct progesterone receptor.

Recent studies have shown that saturable high-capacity nuclear binding sites (termed acceptor sites) for the avian oviduct progesterone receptor can be reconstituted by rehybridizing a specific oviduct chromatin protein fraction (CP-3) to pure hen DNA to generate a reconstituted nucleoacidic protein (NAP). Only a limited number of acceptor sites can be generated on hen DNA even at high protein/DNA ratios. This suggests the existence of a limited number of specific sequences in the avian genome that can participate in the acceptor sites. The studies presented in this paper show a specificity as to the source of DNA that can generate acceptor sites using hen oviduct CP-3 protein. The acceptor protein binds to all DNAs but generates acceptor sites only on DNAs from certain animals. The acceptor sites for the progesterone receptor, generated with heterologous mammalian DNAs and the avian oviduct CP-3 fraction, show saturation not only in number of acceptor sites generated on the DNAs but also in progesterone receptor binding. Binding to these sites is also receptor dependent. Using oviduct receptors from particular physiological states of the birds wherein the receptors do not bind to nuclear sites in vivo, it was found that the cell-free binding to these heterologous complexes of hen CP-3 protein and DNA from another species, termed heterologous NAP, is similarly absent. Thus, the cell-free binding to the native oviduct NAP and the heterologous NAP markedly resembles the nuclear binding in vivo. Interestingly, synthetic DNAs rich in adenine and thymine, but not those rich in guanine and cytosine, are capable of generating acceptor sites. Species-specific DNA sequences, as well as specific chromatin proteins, therefore, appear to be involved in the nuclear acceptor sites for the avian oviduct progesterone receptor. The DNA sequences appear to be conserved throughout most of the vertebrates but not among nonvertebrates as are the steroid hormones and their receptors. The exact numbers and distributions of these sequences in the avian genome are not known.     

RNA polymerase binding sites in λplac5 DNA

The in vitro binding of the Escherichia coli RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) to fragments of lambdaplac5 DNA generated by restriction endonucleases HindII and HindIII has been studied by a filter binding technique. The results are consistent with RNA polymerase binding at p(R)', the INT promoter (p(I)), several sites in the b2 region, the mis promoter, the oop promoter (or p(O)), and p(rm). Binding was also observed on some fragments that are not known to contain active promoters, including the fragment from the cIII-t(L) region. Some of these binding reactions might also be explained by interaction of RNA polymerase with termination sites. Additional polymerase binding sites have been detected by examining which HindII and HindIII sites were not cleaved when digestion was performed after RNA polymerase had been bound to the DNA. This technique revealed polymerase binding at p(L), at p(R), at a site between R and cos, and at a site at the junction of the gamma and cIII-t(L) fragments. A comparison of the location of polymerase binding fragments with the partial denaturation map of the lambda genome indicates that RNA polymerase binding sites are located within A-T rich regions. It is suggested that RNA polymerase binding is a function both of specific sequences (where recognition occurs) and of the base composition of the surrounding regions (which affects the stability of the helix at the specific site).     

Nucleoprotein complex formed between herpes simplex virus UL9 protein and the origin of DNA replication: inter- and intramolecular interactions.

The UL9 gene of herpes simplex virus type 1 encodes an origin-binding protein. UL9 protein purified from baculovirus vector-infected insect cells forms a stable complex with DNA containing the herpes simplex virus origin of DNA replication, oriS. Contained within oriS are two UL9 protein-binding sites, I and II, bracketing an (A + T)-rich region. UL9 protein, visualized by electron microscopy, binds selectively at the site of the origin and covers approximately 120 base pairs. Upon formation of the nucleoprotein complex, the apparent contour length of the DNA is shortened, suggesting that this amount of DNA is wrapped or condensed by the protein. A nucleoprotein complex of similar size and structure forms on an inactive origin deleted for binding site II. Multiple intermolecular interactions occur. In particular, UL9 nucleoprotein complexes interact in trans with other UL9 nucleoprotein complexes such that dimer DNA molecules are formed with a junction at the position of protein binding. The DNA molecules in these intermolecular complexes are aligned predominantly in a parallel orientation.     

Sequence-specific recognition of DNA by zinc-finger peptides derived from the transcription factor Sp1.

We have overexpressed and purified two peptide fragments of Sp1 that contain the three "zinc-finger" domains necessary for specific Sp1 DNA binding. These peptides assume a stable, folded conformation in solution in the presence of Zn2+ as shown by DNA binding assays and NMR spectroscopy. Mobility-shift assays demonstrate that the Sp1 peptides recognize a number of different Sp1 DNA binding sites (GC boxes, with the core sequence GGGCGG). The dissociation constant for a 92-amino acid peptide binding to the GGGGCGGGGC sequence (Kd approximately 10 nM) and the relative affinities for several other DNA sequences definitively demonstrate Sp1-like binding properties. The thermodynamic binding site for Sp1-Zn92 has been mapped using the primer-extension/mobility-shift assay revealing that the 5' portion of the GC box DNA sequence (GGG GCG) contributes more strongly to the total binding energy than the 3' portion (GGGC). These findings are interpreted in the context of the Sp1 amino acid sequence in comparison with the structurally characterized Zif-268/DNA complex. A model is proposed that offers a structural explanation for the ability of Sp1 to recognize a diverse array of DNA sequences in terms of the individual (and different) DNA binding properties of each of the three zinc-finger domains.