In vitro methylation of the 5'-flanking regions of the mouse beta-globin gene

The enzymatic methylation of the 5'-flanking region of the mouse beta-globin (major) gene containing putative regulatory regions has been investigated. In vitro methylation of this 368-base pair regulatory DNA by a DNA methyltransferase obtained from mouse erythroleukemia cells yields an asymmetric methylation pattern. Of the 10 available CG pairs, only 5-6 are modified, leading to one hemimethylated site and two apparently fully methylated sites. Only CG pairs which are localized in a 29-base pair cluster are methylated. The data suggest that a CG cluster approximately 100 base pairs upstream from the CAP site may be the in vivo site of methylation in the 5'-regulator region of the mouse beta-globin gene.     

Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: recognition of pyrimidine-purine and purine-purine steps

The catabolite activator protein (CAP) bends DNA in the CAP-DNA complex, typically introducing a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site, 5'-A1A2A3T4G5T6G7A8T9C10T11 -3' ("primary kink"). In previous work, we showed that CAP recognizes the nucleotide immediately 5' to the primary-kink site, T6, through an "indirect-readout" mechanism involving sequence effects on energetics of primary-kink formation. Here, to understand further this example of indirect readout, we have determined crystal structures of CAP-DNA complexes containing each possible nucleotide at position 6. The structures show that CAP can introduce a DNA kink at the primary-kink site with any nucleotide at position 6. The DNA kink is sharp with the consensus pyrimidine-purine step T6G7 and the non-consensus pyrimidine-purine step C6G7 (roll angles of approximately 42 degrees, twist angles of approximately 16 degrees ), but is much less sharp with the non-consensus purine-purine steps A6G7 and G6G7 (roll angles of approximately 20 degrees, twist angles of approximately 17 degrees). We infer that CAP discriminates between consensus and non-consensus pyrimidine-purine steps at positions 6-7 solely based on differences in the energetics of DNA deformation, but that CAP discriminates between the consensus pyrimidine-purine step and non-consensus purine-purine steps at positions 6-7 both based on differences in the energetics of DNA deformation and based on qualitative differences in DNA deformation. The structures further show that CAP can achieve a similar, approximately 46 degrees per DNA half-site, overall DNA bend through a sharp DNA kink, a less sharp DNA kink, or a smooth DNA bend. Analysis of these and other crystal structures of CAP-DNA complexes indicates that there is a large, approximately 28 degrees per DNA half-site, out-of-plane component of CAP-induced DNA bending in structures not constrained by end-to-end DNA lattice interactions and that lattice contacts involving CAP tend to involve residues in or near biologically functional surfaces.     

Single base-pair mutations in centromere element III cause aberrant chromosome segregation in Saccharomyces cerevisiae.

In this paper we show that a 211-base pair segment of CEN3 DNA is sufficient to confer wild-type centromere function in the yeast Saccharomyces cerevisiae. We used site-directed mutagenesis of the 211-base pair fragment to examine the sequence-specific functional requirements of a conserved 11-base pair segment of centromere DNA, element III (5'-TGATTTATCCGAA-3'). Element III is the most highly conserved of the centromeric DNA sequences, differing by only a single adenine X thymine base pair among the four centromere DNAs sequenced thus far. All of the element III sequences contain specific cytosine X guanine base pairs, including a 5'-CCG-3' arrangement, which we targeted for single cytosine-to-thymine mutations by using sodium bisulfite. The effects of element III mutations on plasmid and chromosome segregation were determined by mitotic stability assays. Conversion of CCG to CTG completely abolished centromere function both in plasmids and in chromosome III, whereas conversion of CCG to TCG decreased plasmid and chromosome stability moderately. The other two guanine X cytosine base pairs in element III could be independently converted to adenine X thymine base pairs without affecting plasmid or chromosome stability. We concluded that while some specific nucleotides within the conserved element III sequence are essential for proper centromere function, other conserved nucleotides can be changed.     

Minimal DNA requirement for topoisomerase II-mediated cleavage in vitro

The minimal DNA requirement for topoisomerase II-mediated DNA cleavage in vitro was determined by analyzing the interaction of the enzyme with sets of DNA substrates varying successively by single bases at the 5'- or 3'-end of either strand. A 16-base pair double-stranded region was established as the minimal duplex region required for topoisomerase II cleavage activity. The region was located symmetrically around the 4-base staggered cleavage site. Topoisomerase II-mediated cleavage within the 16-base pair core duplex, however, required single-stranded regions flanking the duplex to either the 5'- or 3'-sides, or an extension at both ends of the duplex with 1 or more base pairs.     

Structure of the DNA interstrand cross-link of 4,5',8-trimethylpsoralen.

4,5',8-Trimethylpsoralen (TMP) cross-links a 5' TpA or a 5' ApT site by photoreacting with one thymine moiety in each DNA strand. We are interested in whether psoralen interstrand cross-links all share one structure or whether there are significant differences. In this paper, we employed a rapid method for probing the structure of the cross-link by making a series of TMP cross-linked duplexes containing specific base-pair mismatches. The relative stability provided by a base pair can be correlated with neighboring base pairs by comparing the extents of gel retardation when base-pair mismatches happen in each position. From our studies, we infer that with respect to the furan-side strand, the 5'T.A base pair of the two T.A base pairs in the TpA site is not hydrogen bonded. Immediately on each side of the cross-linked TpA site is a highly stabilized base pair. Next, a region of decreased stability occurs in each arm of a cross-linked duplex and these base pairs of least stability are located farther away from the cross-linked thymines as the lengths of the arms of the cross-linked helix increase. Finally, even in 7 M urea at 49 degrees C the cross-linked helix is hydrogen bonded at both ends of a duplex of 22 base pairs. We propose that the structures of interstrand cross-links in DNA vary appreciably with the DNA sequence, the length of the DNA duplex, and the structures of the DNA cross-linking agents.     

DNA sequence recognition by a eukaryotic sequence-specific endonuclease, Endo.SceI, from Saccharomyces cerevisiae

A eukaryotic sequence-specific endonuclease, Endo.SceI, causes sequence-specific double-stranded scission of double-stranded DNA to produce cohesive ends with four bases protruding at the 3' termini. Unlike in the case of restriction enzymes, an asymmetric 26-base pair consensus sequence was found around the cleavage site for Endo.SceI instead of a common sequence. We analyzed the base pairs that interacted with Endo.SceI on the recognition of its cleavage sites. A region comprising -10 through +16 base pairs from the center of the cleavage site was shown to be essential and sufficient for the sequence-specific cutting with Endo.SceI by experiments involving synthesized DNAs. Methylation interference experiments indicate that bases in the region comprising the +7 through +14 base pairs is involved in close contact with Endo.SceI in its recognition of the cleavage site. This +7 through +14-base pair region overlaps the most stringently conserved sequence in the consensus sequence for the cleavage site, suggesting that this region constitutes the core for the recognition by Endo.SceI.     

Recognition of GT mismatches by Vsr mismatch endonuclease

The Vsr mismatch endonuclease recognises the sequence CTWGG (W = A or T) in which the underlined thymine is paired with guanine and nicks the DNA backbone on the 5'-side of the mispaired thymine. By using base analogues of G and T we have explored the functional groups on the mismatch pair which are recognised by the enzyme. Removal of the thymine 5-methyl group causes a 60% reduction in activity, while removing the 2-amino group of guanine reduces cleavage by 90%. Placing 2-amino-purine or nebularine opposite T generates mis-matches which are cut at a much lower rate (0.1%). When either base is removed, generating a pseudoabasic site (1', 2'-dideoxyribose), the enzyme still produces site-specific cleavage, but at only 1% of the original rate. Although TT and CT mismatches at this position are cleaved at a low rate (approximately 1%), mismatches with other bases (such as GA and AC) and Watson-Crick base pairs are not cleaved by the enzyme. There is also no cleavage when the mismatched T is replaced with difluorotoluene.     

Indirect readout of DNA sequence at the primary-kink site in the CAP-DNA complex: DNA binding specificity based on energetics of DNA kinking.

The catabolite activator protein (CAP) makes no direct contact with the consensus base-pair T:A at position 6 of the DNA half-site 5'-A(1)A(2)A(3)T(4)G(5)T(6)G(7)A(8)T(9)C(10)T(11)-3' but, nevertheless, exhibits strong specificity for T:A at position 6. Binding of CAP results in formation of a sharp DNA kink, with a roll angle of approximately 40 degrees and a twist angle of approximately 20 degrees, between positions 6 and 7 of the DNA half-site. The consensus base-pair T:A at position 6 and the consensus base-pair G:C at position 7 form a T:A/G:C step, which is known to be associated with DNA flexibility. It has been proposed that specificity for T:A at position 6 is a consequence of formation of the DNA kink between positions 6 and 7, and of effects of the T:A(6)/G:C(7) step on the geometry of DNA kinking, or the energetics of DNA kinking. In this work, we determine crystallographic structures of CAP-DNA complexes having the consensus base-pair T:A at position 6 or the non-consensus base-pair C:G at position 6. We show that complexes containing T:A or C:G at position 6 exhibit similar overall DNA bend angles and local geometries of DNA kinking. We infer that indirect readout in this system does not involve differences in the geometry of DNA kinking but, rather, solely differences in the energetics of DNA kinking. We further infer that the main determinant of DNA conformation in this system is protein-DNA interaction, and not DNA sequence.     

Characterization of the herpes simplex virus origin binding protein interaction with OriS.

The origin binding protein (OBP) of herpes simplex virus (HSV), which is essential for viral DNA replication, binds specifically to sequences within the viral replication origin(s) (for a review, see Challberg, M.D., and Kelly, T. J. (1989) Annu. Rev. Biochem. 58, 671-717). Using either a COOH-terminal OBP protein A fusion or the full-length protein, each expressed in Escherichia coli, we investigated the interaction of OBP with one HSV origin, OriS. Binding of OBP to a set of binding site variant sequences demonstrates that the 10-base pair sequence, 5' CGTTCGCACT 3', comprises the OBP-binding site. This sequence must be presented in the context of at least 15 total base pairs for high affinity binding, Ka = approximately 0.3 nM. Single base pair mutations in the central CGC sequence lower the affinity by several orders of magnitude, whereas a substitution at any of the other seven positions reduces the affinity by 10-fold or less. OBP binds with high affinity to duplex DNA containing mismatched base pairs. This property is exploited to analyze OBP binding to DNA heteroduplexes containing singly substituted mutant and wild-type DNA strands. For positions 2, 3, 5, 6, 7, 8, and 9, substitutions are tolerated on one or the other DNA strand, indicating that base-mediated interactions are limited to one base of each pair. For both Boxes I and II, these interactions are localized to one face of the DNA helix, forming a recognition surface in the major groove. In OriS, the 31 base pairs which separate Boxes I and II orient the two interaction surfaces to the same side of the DNA.     

Consensus DNA site for the Escherichia coli catabolite gene activator protein (CAP): CAP exhibits a 450-fold higher affinity for the consensus DNA site than for the E. coli lac DNA site.

We have synthesized two 40 base pair DNA fragments; one fragment contains the consensus DNA site for CAP (fragment 'ICAP'); the other fragment contains the E. coli lac promoter DNA site for CAP (fragment 'LCAP'). We have investigated the binding of CAP to the two DNA fragments using the nitrocellulose filter binding assay. Under standard conditions [( NaCl] = 200 mM, pH = 7.3), CAP exhibits a 450-fold higher affinity for ICAP than for LCAP. The salt dependence of the binding equilibrium indicates that CAP makes eight ion pairs with ICAP, but only six ion pairs with LCAP. Approximately half of the difference in binding free energy for interaction of CAP with ICAP vs. LCAP is attributable to this difference in ion-pair formation. The pH dependence of the binding equilibrium indicates that the eight CAP-ICAP ion pairs and the six CAP-LCAP ion pairs do not involve His residues of CAP.     

Human methylated DNA-binding protein. Determinants of a pBR322 recognition site

Methylated DNA-binding protein (MDBP) from human placenta has a high affinity for a site in pBR322 (pB site 1) when that site is methylated at its CpG dinucleotides. Dimethyl sulfate interference analysis and experiments with ligands prepared by oligonucleotide-directed mutagenesis indicate that 15 contiguous base pairs, 14 of which exhibit hyphenated dyad symmetry, influence MDBP binding to pB site 1. These 14 base pairs, 5'-RTMGYCAMGG(M/T)GAY-3' (M, 5-methylcytosine), suffice for recognition by MDBP as demonstrated with a double-stranded, MpG-containing oligonucleotide used as a free ligand or cloned into M13mp19 and subsequently methylated. Seven single-site mutations at different positions of this 14-base pair region largely eliminated binding, and several others increased binding up to 2-fold when compared to the nonmutant, triply methylated sequence. However, MDBP recognizes a site in hemimethylated M13mp19 replicative form DNA, which was homology to pB site 1 at only 10 of 14 base pairs, and all four of these different base pairs are equivalent to transversions. Based upon the above data, a mixed oligonucleotide probe was constructed that contains variants of pB site 1 which should be recognized by MDBP. This 14-base probe hybridizes under stringent conditions to a number of discrete fragments in restriction digests of human DNA. this suggests that there are multiple pB site 1-related sequences in human DNA that might, when methylated, bind MDBP in vivo.     

Contacts between the factor TUF and RPG sequences

The yeast TUF factor binds specifically to RPG-like sequences involved in multiple functions at enhancers, silencers, and telomeres. We have characterized the interaction of TUF with its optimal binding sequence, rpg-1 (1-ACACCCATACATTT-14), using a gel DNA-binding assay in combination with methylation protection and mutagenesis experiments. As many as 10 base pairs appear to be engaged in factor binding. Analysis of a collection of 30 different RPG mutants demonstrated the importance of 8 base pairs at position 2, 3, 4, 5, 6, 7, 10, and 12 and the critical role of the central GC pair at position 5. Methylation protection data on four different natural sites confirmed a close contact at positions 4, 5, 6, and 10 and suggested additional contacts at base pairs 8, 12, and 13. The derived consensus sequence was RCAAYCCRYNCAYY. A quantitative band shift analysis was used to determine the equilibrium dissociation constant for the complex of TUF and its optimal binding site rpg-1. The specific dissociation constant (K8) was found to be 1.3 x 10(-11) M. The comparison of the K8 value with the dissociation constant obtained for nonspecific DNA sites (Kn8 = 8.7 x 10(-6) M) shows the high binding selectivity of TUF for its specific RPG target.     

Construction of DNA substrates modified with psoralen at a unique site and study of the action mechanism of ABC excinuclease on these uniformly modified substrates

Psoralens bind to DNA noncovalently and upon exposure to near UV (320-400 nm) light produce covalent adducts. Thymidine residues in DNA, especially those at 5'-TpA-3' sequences, are most susceptible to the photochemical reaction. This property of the reaction and the recent advances in oligonucleotide synthesis and separation has enabled us to construct DNA fragments containing psoralen adducts at a specific site. The octanucleotide 5'-TCGTAGCT-3' was photoreacted (in the presence of the complementary strand) with the synthetic psoralen 4'-hydroxymethyl-4,5',8-trimethylpsoralen to obtain oligonucleotides adducted via the furan or pyrone ring at the internal thymine. These modified octanucleotides were ligated to nonmodified oligonucleotides to obtain a 40-base pair DNA fragment containing a psoralen adduct at a central location. The modified fragment having the thymine-furan side 4'-hydroxymethyl-4,5',8-trimethylpsoralen adduct was irradiated with 360 nm of light to produce an interstrand cross-link, and this cross-linked DNA was purified to homogeneity. These uniquely modified DNAs were used as substrates for Escherichia coli ABC excinuclease to determine its incision mechanism unambiguously and to determine the contact sites of the enzyme. ABC excinuclease mediates the cleavage of the 8th and 5th phosphodiester bonds 5' and 3', respectively, to psoralen monoadducts, and the 9th (5') and 3rd (3') phosphodiester bonds to the furan-side thymine of the cross-link. Preliminary DNaseI footprinting studies show that ABC excinuclease protects the whole 40-base pair fragment from DNaseI, and binding of the A and B subunits to the furan side-monoadducted substrate produces two hypersensitive phosphodiester bonds in the vicinity of the 5' incision site of ABC excinuclease.     

Specific contacts between the FLP protein of the yeast 2-micron plasmid and its recombination site

Contact points between the FLP protein of the yeast 2-micron plasmid and its recombination site have been defined. Important features of the region previously defined as the minimal recombination site in vitro include a pair of 13-base pair inverted repeats separated by an 8-base pair spacer. The two FLP protein-binding sites within this region are 12 base pairs in length. In each case they include the internal 11 base pairs of one of the 13-base pair repeats, as well as the adjacent base pair within the spacer. The internal 6 base pairs within the spacer are not involved in binding or recognition by FLP protein. When the size of the spacer is increased or decreased by one base pair, the distance between the cleavage points is also increased or decreased correspondingly by one base pair. Points of cleavage are unaffected by changes in the spacer sequence. Specific contact points involving purine residues, identified by methylation protection and recombination interference experiments, are located in both the major and minor grooves of the DNA. Additional contact points between FLP protein and phosphate groups in the phosphate-deoxyribose backbone are clustered near the cleavage sites.     

Novel insertion mutation in Caenorhabditis elegans.

The mutation e1662 is an allele of the Caenorhabditis elegans unc-54 gene induced with the difunctional alkylating agent 1,2,7,8-diepoxyoctane. unc-54 encodes the major myosin heavy chain isozyme of body wall muscle cells. Filter-transfer hybridization and DNA sequence analysis show that e1662 is an insertion of 288 base pairs of DNA within unc-54. The inserted DNA is identical to a 288-base pair region of unc-54 located ca. 600 base pairs from the insertion site. Thus, e1662 is a displaced duplication. A 14-base pair sequence located at one end of the duplicated segment is found adjacent to the site of insertion. These homologous sequences are juxtaposed head-to-tail by the insertion event. e1662 thus contains a tandem direct repeat extending across one of its junctions.     

Three additional operators, Op21, Op68, and Op88, of bacteriophage P1. Evidence for control of the P1 dam methylase by Op68

The repressor of bacteriophage P1, encoded by the c1 gene, is responsible for maintaining the P1 prophage in the lysogenic state. Previously, 11 c1 repressor binding sites or operators scattered over the whole genome of P1 have been found. From sequence analysis an asymmetric, 17-base pair consensus sequence, ATTGCTCTAATAAATTT, was derived. Using a synthetic 15-base-long oligodeoxyribonucleotide as operator probe, we have identified three additional operators. We have mapped the operators at the positions 21,68, and 88 of the P1 genome and determined their sequence. These operators are controlled by c1 because corresponding P1 DNA fragments (i) require c1 repressor in vivo in order to be clonable in multicopy plasmids, (ii) exhibit a c1-repressible promoter activity, (iii) are retarded by c1 repressor protein during electrophoresis, and (iv) contain the 17-base pair consensus sequence with one mismatch base each. Furthermore, we suggest that expression of the DNA adenine methylase (dam) encoded by P1 is controlled via Op68.