Analysis of nuclear factor I binding to DNA using degenerate oligonucleotides.

Nuclear factor I (NFI) binds tightly to DNA containing the consensus sequence TGG(N)6-7GCCAA. To study the role of the spacing between the TGG and GCCAA motifs, oligonucleotides homologous to the NFI binding site FIB-2 were synthesized and used for binding assays in vitro. The wild-type site (FIB-2.6) has a 6bp spacer region and binds tightly to NFI. When the size of this spacer was altered by +/- 1 or 2bp the binding to NFI was abolished. To further assess the role of the spacer and bases flanking the motifs, two oligonucleotide libraries were synthesized. Each member of these libraries had intact TGG and GCCAA motifs, but the sequence of the spacer and the 3bp next to each motif was degenerate. The library with a 6bp spacer bound to NFI to 40-50% the level of FIB-2.6. The library with a 7bp spacer bound to NFI to only 4% the level of FIB-2.6 and some of this binding was weaker than that of FIB-2.6 DNA. This novel use of degenerate DNA libraries has shown that: 1) the structural requirements for FIB sites with a 7bp spacer are more stringent than for sites with a 6bp spacer and 2) a limited number of DNA structural features can prevent the binding of NFI to sites with intact motifs and a 6bp spacer region.     

Enhancement of DNA replication by transcription factors NFI and NFIII/Oct-1 depends critically on the positions of their binding sites in the adenovirus origin of replication

The origin of DNA replication of many human adenoviruses is composed of a highly conserved core origin and an auxiliary region, containing the binding sites for NFI and NFIII/Oct-1. We examined enhancement of DNA replication in vitro by the purified functional DNA-binding domains of NFI (NFI-BD) and NFIII/Oct-1 (the POU domain), using origins in which the positions of the binding sites for these proteins were transposed. Insertion or deletion of two or three base pairs between the core origin and the NFI binding site resulted in a 3-5-fold decrease of stimulation, whereas larger insertions gradually reduced the stimulation further. Mutants in which the NFI binding site was separated approximately one or two helical turns from the core origin by AT-rich sequences could still be stimulated by NFI. In contrast, insertion of two or more base pairs between the NFI and NFIII/Oct-1 binding sites abolished stimulation by NFIII/Oct-1 almost completely. Furthermore, stimulation by this protein was lost when the Ad2 NFIII/Oct-1 binding site was transposed to a position closer to the core origin, destroying the NFI binding site. This shows that the position of the NFIII/Oct-1 binding site is essential for stimulation. Models to explain these position-dependent effects on stimulation are discussed.     

A consensus DNA-binding site for the androgen receptor.

We have used a DNA-binding site selection assay to determine a consensus binding sequence for the androgen receptor (AR). A purified fusion protein containing the AR DNA-binding domain was incubated with a pool of random sequence oligonucleotides, and complexes were isolated by gel mobility shift assays. Individually selected sites were characterised by nucleotide sequencing and compiled to give a consensus AR-binding element. This sequence is comprised of two 6-basepair (bp) asymmetrical elements separated by a 3-bp spacer, 5'-GGA/TACANNNTGTTCT-3', similar to that described for the glucocorticoid response element. Inspection of the consensus revealed a slight preference for G or A nucleotides at the +1 position in the spacer and for A and T nucleotides in the 3'-flanking region. Therefore, a series of oligonucleotides was designed in which the spacer and flanking nucleotides were changed to the least preferred sequence. Competition experiments with these oligonucleotides and the AR fusion protein indicated that an oligonucleotide with both the spacer and flanking sequences changed had greater than 3-fold less affinity than the consensus sequence. The functional activity of these oligonucleotides was also assessed by placing them up-stream of a reporter gene in a transient transfection assay and correlated with the affinity with which the AR fusion protein bound to DNA. Therefore, sequences surrounding the two 6-bp half-sites influence both the binding affinity for the receptor and the functional activity of the response element.     

Chemical probe and missing nucleoside analysis of Flp recombinase bound to the recombination target sequence.

The Flp protein catalyzes a site-specific recombination reaction between two 47 bp DNA sites without the assistance of any other protein or cofactor. The Flp recognition target (FRT) site consists of three nearly identical sequences, two of which are separated by an 8 bp spacer sequence. In order to gain insight into this remarkable protein-DNA interaction we used a variety of chemical probe methods and the missing nucleoside experiment to examine Flp binding. Hydroxyl radical footprints of Flp bound to a recombinationally-competent site fall on opposite faces of canonical B-DNA. The 8 bp spacer region between the two Flp binding sites becomes reactive towards 5-phenyl-1,10-phenanthroline.copper upon Flp binding, indicating that once bound by Flp, this segment of DNA is not in the B-form. Missing nucleoside analysis reveals that within each binding site the presence of two nucleosides on the top strand and four on the bottom, are required for formation of a fully-occupied FRT site. In contrast, loss of any nucleoside in the three binding sites in the FRT interferes with formation of lower-occupancy complexes. DNA molecules with gaps in the 8 bp spacer region are over-represented in complexes with either two or three binding sites occupied by Flp, evidence that DNA flexibility facilitates the cooperative interaction of Flp protomers bound to a recombinationally-active site.     

The FLP recombinase of the 2 micron circle DNA of yeast: interaction with its target sequences

We have studied the interaction of purified FLP protein with restriction fragments from the substrate 2mu circle DNA of yeast. We find that FLP protects about 50 bp of DNA from nonspecific nuclease digestion. The protected site consists of two 13 bp inverted repeat sequences separated by an 8 bp spacer region. A third 13 bp element is also protected by binding of the FLP protein. We demonstrate that FLP introduces single- and double-strand breaks into the substrate DNA. This site-specific cleavage occurs at the margins of the spacer region, generating 8 bp 5' protruding ends with 5'-OH and 3'-protein-bound termini. Binding to mutant sites and half-sites demonstrates that the third symmetry element is not important for binding and cleavage by the FLP protein. The integrity of the core region is important for the cleavage activity of FLP.     

Nucleotide sequence and LexA regulation of the Escherichia coli recN gene.

The nucleotide sequence of a 2224 bp region of the Escherichia coli chromosome that carries the LexA regulated recN gene has been determined. A region of 1701 nucleotides encoding a polypeptide of 567 amino acids with a predicted molecular weight of 63,599 was identified as the most probable sequence for the recN structural gene. The proposed initiation codon is preceded by a reasonable Shine-Dalgarno sequence and a promoter region containing two 16 bp sequences, separated by 6 bp, that match the consensus sequence (SOS box) for binding LexA protein. DNA fragments containing this putative promoter region are shown to bind LexA in vitro and to have LexA-regulated promoter activity in vivo. The amino acid sequence of RecN predicted from the DNA contains a region that is homologous to highly conserved sequences found in several DNA repair enzymes and other proteins that bind ATP. A sequence of 9 amino acids was found to be homologous to a region of the RecA protein of E. coli postulated to have a role in DNA/nucleotide binding.     

Numerous nuclear proteins bind the long control region of human papillomavirus type 16: a subset of 6 of 23 DNase I-protected segments coincides with the location of the cell-type-specific enhancer.

The long control region of the human papillomavirus type 16 genome is 856 base pairs (bp) long. It contains a cell-type-specific enhancer, a glucocorticoid response element, and sequences mediating the response to the viral gene products of open reading frame E2; all three regulate the promoter P97. We mapped binding sites of trans-acting proteins relevant for the cell-type-specific enhancer and other cis-acting elements by DNase I footprint experiments with nuclear extracts from HeLa cells. Throughout the human papillomavirus type 16 long control region 23 footprints protect 557 of 900 bp. Nine footprints fall into a 400-bp segment that was previously identified to contain the cell-type-specific enhancer. Variations of the protein concentration in the footprint reaction do not affect six of these nine footprints. At high protein concentrations, three footprints fuse to a 106-bp protected region, suggesting that this segment specifically binds several proteins of lower affinity or abundance. Unexpectedly, extracts from human MCF7 and mouse 3T3 cells, in which the enhancer is inactive, give footprints identical to those obtained with HeLa extracts. Seven footprints contain the sequence 5'-TTGGC-3'. Footprint competition experiments suggest that factor NFI binds to these seven motifs. Competition with cloned oligonucleotides in transfections suggests that these elements contribute to the enhancer function. Subcloning identifies a 232-bp fragment between positions 7524 and 7755 as sufficient for full enhancer activity. Several of the six footprinted elements on this segment may cooperate functionally, since subclones of this region show decreased or no cell-type-specific enhancer function.     

Sequence analysis of the 5' non coding region of Turkish HCV isolates: implications for PCR diagnosis

Hepatitis C virus (HCV) is a positive-strand RNA virus related to pestiviruses and flaviviruses. The 5' noncoding region (NCR) of the virus genome consists of 324-341 nucleotides and is generally highly conserved among different HCV isolates which has made this region the choice for primer selection in amplification of HCV sequences by polymerase chain reaction (PCR). In this study, we report the partial nucleotide sequences of the 5'-NCR from type 1a (n = 4), type 1b (n = 6) and type 4 (n = 1) Turkish HCV isolates. Sequence information was obtained by direct sequencing of RT-PCR product using biotinylated primers and single strands were sequenced using T7 DNA polymerase after binding to streptavidin coated magnetic beads. In comparison to prototype type 1a consensus sequence, all type 1b sequences had A-G substitution at position - 99. Nucleotid changes from the prototype 1a sequence were found in 12 of the 174 nucleotide positions. The most variable domain spans 51 nucleotides (positions - 167 to - 117) where nine polymorphic sites were identified. Although the nucleotide sequence of the 5'-noncoding region is highly conserved there are type-specific polymorphic sites within this region that has to be taken into consideration in the design of oligonucleotide primers for reliable amplification of sequences from different HCV genotypes.     

Sequence Characterization of a Unique Intergenic Spacer in Gadiformes Mitochondrial DNA

The nucleotide sequences of intergenic spacers located between the tRNA^Thr and tRNA^Pro genes in mitochondrial DNA of cod fishes (order Godiformes) were determined. Spacers from eight species representing two families of cod fishes were analyzed and found to vary in size from 25 to 99 bp. Each spacer sequence contains one or two copies of a conserved 17-bp motif. Four to five central nucleotides of this motif constitute a substitutional hot spot as observed from interspecific and intraspecific comparisons. The substitution rate of the spacer is approximately twice that of the variable part I of the mitochondrial DNA control region, making this sequence region interesting as a molecular marker in population studies or stock assessments of cod fishes. We propose that the spacer originated in a duplication event and evolved into a functional domain, perhaps by binding regulatory proteins. Accepted February 26, 1999     

Protein:DNA interactions at chromosomal loop attachment sites

We have recently identified an evolutionarily conserved class of sequences that organize chromosomal loops in the interphase nucleus, which we have termed "matrix association regions" (MARs). MARs are about 200 bp long, AT-rich, contain topoisomerase II consensus sequences and other AT-rich sequence motifs, often reside near cis-acting regulatory sequences, and their binding sites are abundant (greater than 10,000 per mammalian nucleus). Here we demonstrate that the interactions between the mouse kappa immunoglobulin gene MAR and topoisomerase II or the "nuclear matrix" occur between multiple and sometimes overlapping binding sites. Interestingly, the sites most susceptible to topoisomerase II cleavage are localized near the breakpoints of a previously described illegitimate recombination event. The presence of multiple binding sites within single MARs may allow DNA and RNA polymerase passage without disrupting primary loop organization.     

Expression of the mitochondrial genome in HeLa cells : XIV. The relative positions of the 4 s RNA genes and of the ribosomal RNA genes in mitochondrial DNA

HeLa mitochondrial 4 s RNA has been covalently coupled to the electron opaque label, ferritin, which is visible in the electron microscope. Mixtures of HeLa mitochondrial 12 s ribosomal RNA, 16 s rRNA and/or the 4 s RNA-ferritin conjugate have been hybridized to separated heavy (H) and light (L) strands of HeLa mitochondrial DNA, or to a mixture of H and L strands. The relative positions of the duplex regions corresponding to the 12 s and 16 s rRNA—DNA hybrids and of the ferritin-labeled 4 s RNA's have been mapped in the electron microscope after spreading the DNA strands by the formamide modification of the basic protein film technique. The 12 s and 16 s duplex regions have lengths of 0·-26 ± 0.04 μm and 0.46 ± 0.07 μm, respectively. They are separated by a single-strand region of length 0.047 ± 0.017 μm, corresponding to 160 ± 60 nucleotides. There are nine reproducible binding sites for 4 s RNA on the H strand. One such site lies within the spacer region between the 12 s and 16 s coding sequences, one site is immediately adjacent to the other side of the 12 s sequence and one is adjacent to the other side of the 16 s sequence. The other 4 s sites are rather evenly spaced along the DNA strand of total length 15,600 nucleotides, except that two of them are clustered with a spacing of 120 ± 30 nucleotides between them. There are three 4 s RNA coding sequences on the L strand, separated from one another by 2280 and 3900 nucleotides, respectively.     

Structure and function of 59-base element recombination sites associated with mobile gene cassettes

The integration of gene cassettes into integrons is effected by site-specific recombination catalysed by an integrase, IntI, encoded by the integron. The cassette-associated recombination sites, 59-base elements, are not highly conserved and vary in length from 57 to 141 bp. They can be identified by their location and the relationship of over 20 bp at their outer ends to consensus sequences that are imperfect inverted repeats of one another. The recombination cross-over occurs close to one end of the 59-base element, within a conserved core site with the consensus sequence GTTAGGC or GTTRRRY. By introducing single-base changes at each of these positions in the aadB 59-base element, bases that are critical for site activity were identified. The recombination cross-over was also localized to a unique position between the adjacent G and T residues. Changes introduced in the conserved AAC of the inverse core site (GCCTAAC or RYYYAAC) located at the opposite end of the 59-base element also reduced site activity but to a lesser extent. Sequences of rare recombinants revealed an alternative position for strand exchange and led to the conclusion that 59-base elements comprise two simple sites, analogous to those recognized by other integrases, with each simple site made up of a pair of inversely oriented IntI binding domains separated by a spacer of 7 or 8 bp. Re-examination of the sequences of all known 59-base elements revealed that this simple site configuration was present at both the left and right ends in all 59-base elements. The identity of bases in the spacer is not required for efficient recombination and the cross-over is located at one end of the spacer, suggesting that during IntI1-mediated recombination only one strand exchange occurs.     

Mutations in the spacer region of Drosophila mitochondrial DNA polymerase affect DNA binding, processivity, and the balance between Pol and Exo function

The catalytic subunit (alpha) of mitochondrial DNA polymerase (pol gamma) shares conserved DNA polymerase and 3'-5' exonuclease active site motifs with Escherichia coli DNA polymerase I and bacteriophage T7 DNA polymerase. A major difference between the prokaryotic and mitochondrial proteins is the size and sequence of the region between the exonuclease and DNA polymerase domains, referred to as the spacer in pol gamma-alpha. Four gamma-specific conserved sequence elements are located within the spacer region of the catalytic subunit in eukaryotic species from yeast to humans. To elucidate the functional roles of the spacer region, we pursued deletion and site-directed mutagenesis of Drosophila pol gamma. Mutant proteins were expressed from baculovirus constructs in insect cells, purified to near homogeneity, and analyzed biochemically. We find that mutations in three of the four conserved sequence elements within the spacer alter enzyme activity, processivity, and/or DNA binding affinity. In addition, several mutations affect differentially DNA polymerase and exonuclease activity and/or functional interactions with mitochondrial single-stranded DNA-binding protein. Based on these results and crystallographic evidence showing that the template-primer binds in a cleft between the exonuclease and DNA polymerase domains in family A DNA polymerases, we propose that conserved sequences within the spacer of pol gamma may position the substrate with respect to the enzyme catalytic domains.     

The origin of adenovirus DNA replication: minimal DNA sequence requirement in vivo.

Adenovirus mini-chromosomes which contain two cloned, inverted adenovirus termini replicate in vivo when supplied with non-defective adenovirus as a helper. This system has been used to define the minimum cis acting DNA sequences required for adenovirus DNA replication in vivo. Deletions into each end of the adenovirus inverted terminal repeat (ITR) were generated with Bal31 exonuclease and the resulting molecules constructed into plasmids which contained two inverted copies of the deleted ITR separated by the bacterial neomycin phosphotransferase gene. To determine the effect of the deletion in vivo plasmids cleaved to expose the adenovirus termini were co-transfected with adenovirus type 2 DNA into tissue culture cells. The replicative ability of the molecules bearing adenovirus termini was assayed by Southern blotting of extracted DNA which had been treated with DpnI, a restriction enzyme which cleaves only methylated and therefore unreplicated, input DNA. Molecules containing the terminal 45 bp of the viral genome were fully active whereas molecules containing only 36 bp were in-active in this assay. Therefore sequences required for DNA replication are contained entirely within the terminal 45 bp of the viral genome. Thus, both the previously described highly conserved region (nucleotides 9-18) and the binding site for the cellular nuclear factor I (nucleotides 19-48) are essential for adenovirus DNA replication in vivo.