Identification and purification of DBF-A, a double-stranded DNA-binding protein from Saccharomyces cerevisiae.

Using oligonucleotide affinity chromatography with DNase I footprinting as an assay we have looked for proteins that interact with sequence elements within the yeast origin of replication, autonomously replicating sequence 1 (ARS1). In this work we describe a protein that binds with high affinity to DNA but displays only moderate sequence specificity. It is eluted at 0.7 M salt from an ARS1 oligonucleotide column. Footprinting analysis on ARS1 at a high protein concentration revealed at least three sites of protection flanking element A and its repeats. Element A itself is rendered hypersensitive to DNase I digestion upon protein binding. This pattern is also observed for the H4 and HMR-E ARSs, suggesting that the protein alters the DNA conformation at element A and its repeats. The affinity-purified fraction is also capable of supercoiling a relaxed, covalently closed plasmid in the presence of topoisomerase. Highly purified preparations of the protein are enriched in an 18-kDa polypeptide which can be renatured from a denaturing gel and shown to bind ARS1 DNA. We have designated this protein DBF-A, DNA-binding factor A.     

Purification and characterization of a protein that binds to the recombination signal sequence of the immunoglobulin J kappa segment.

A protein that binds to the recombination signal sequence (RS) of the immunoglobulin J kappa segment was purified almost to homogeneity from the nuclear extract of a murine pre-B cell line 38B9. A similar binding protein was found in lymphoid cell lines but not in non-lymphoid cell lines. The binding activity was associated with a polypeptide with a molecular weight of 60,000. DNase I footprinting analysis demonstrated that this binding protein interacted with the heptamer and several 3' bases close to the heptamer. The Kd value of the J kappa RS binding protein to the J kappa RS was 1 nM. One base substitution in the heptamer of the J kappa RS greatly reduced the affinity of the J kappa RS binding protein. The high specificity of the binding site of the J kappa RS binding protein suggests that this protein may be involved in V-J recombination.     

Structure of the chromosomal copy of yeast ARS1

We have used deoxyribonuclease I (DNase I) and methidium-propyl-EDTA.Fe(II) digestion to characterize the chromosomal structure of the single-copy autonomously replicating sequence ARS1. The major feature of this chromatin is a region of strong hypersensitivity to both cleavage agents. The hypersensitive region contains most of the DNA sequences which have been suggested by in vitro mutagenesis studies [Celniker, S., Sweder, K., Srienc, F., Bailey, J., & Campbell, J. (1984) Mol. Cell. Biol. 4, 2455-2466] to be important in ARS function. It lies at the downstream end of the TRP1 gene. A chromosomal DNase I footprinting analysis was carried out on the hypersensitive region. These data give direct evidence for several localized DNA/protein contacts within the hypersensitive region. The most prominent of these chromatin-dependent contacts is located on the functionally most important 11 base pairs of ARS DNA. On the TRP1 side of the hypersensitive region, there are positioned nucleosomes. On the other side of the hypersensitive region, there is a complex (and possibly heterogeneous) structure.     

A single-stranded DNA binding protein from S. cerevisiae specifically recognizes the T-rich strand of the core sequence of ARS elements and discriminates against mutant sequences.

A protein named ssARS-T binding protein has been purified from yeast that specifically binds to the T-rich strand of the consensus core sequence of yeast autonomously replicating sequence (ARS) elements. As assayed from gel mobility shift experiments the ssARS-T protein shows characteristics of a sequence specific single-stranded DNA binding protein. The complementary A-rich strand of the ARS core sequence is bound much more weakly and no binding can be detected for the double-stranded form of the core sequence. Three single base substitutions in the core sequence that are known to abolish ARS function in vivo also lead to weaker binding of the core sequence to the ssARS-T protein in vitro. The strong correlation between the binding of mutated sequences in vitro and the ARS properties of these sequences in vivo points to an essential function of the ssARS-T protein during replication initiation in yeast ARS elements.     

ARS binding factor I of the yeast Saccharomyces cerevisiae binds to sequences in telomeric and nontelomeric autonomously replicating sequences.

We have analyzed various autonomously replicating sequences (ARSs) in yeast nuclear extract with ARS-specific synthetic oligonucleotides. The EI oligonucleotide sequence, which is derived from HMRE-ARS, and the F1 oligonucleotide sequence, which is derived from telomeric ARS120, appeared to bind to the same cellular factor with high specificity. In addition, each of these oligonucleotides was a competitive inhibitor of the binding of the other. Binding of the ARS binding factor (ABF) to either of these oligonucleotides was inhibited strongly by plasmids containing ARS1 and telomeric TF1-ARS. DNase I footprinting analyses with yeast nuclear extract showed that EI and F1 oligonucleotides eliminated protection of the binding site of ARS binding factor I (ABFI) in domain B of ARS1. Sequence analyses of various telomeric (ARS120 and TF1-ARS) and nontelomeric ARSs (ARS1 and HMRE-ARS) showed the presence of consensus ABFI binding sites in the protein binding domains of all of these ARSs. Consequently, the ABFI and ABFI-like factors bind to these domain B-like sequences in a wide spectrum of ARSs, both telomeric and nontelomeric.     

Quantitative footprinting analysis using a DNA-cleaving metalloporphyrin complex

The results of quantitative footprinting studies involving the antiviral agent netropsin and a DNA-cleaving cationic metalloporphyrin complex are presented. An analysis of the footprinting autoradiographic spot intensities using a model previously applied to footprinting studies involving the enzyme DNase I [Ward, B., Rehfuss, R., Goodisman, J., & Dabrowiak, J. C. (1988) Biochemistry 27, 1198-1205] led to very low values for netropsin binding constants on a restriction fragment from pBR-322 DNA. In this work, we show that, because the porphyrin binds with high specificity to DNA, it does not report site loading information in the same manner as does DNase I. We elucidate a model involving binding equilibria for individual sites and include competitive binding of drug and porphyrin for the same site. The free porphyrin and free drug concentrations are determined by binding equilibria with the carrier (calf thymus DNA) which is present in excess and acts as a buffer for both. Given free porphyrin and free netropsin concentrations for each total drug concentration in a series of footprinting experiments, one can calculate autoradiographic spot intensities in terms of the binding constants of netropsin to the various sites on the 139 base pair restriction fragment. The best values of these binding constants are determined by minimizing the sum of the squared differences between calculated and experimental footprinting autoradiographic spot intensities. Although the determined netropsin binding constants are insensitive to the value assumed for the porphyrin binding constant toward its highest affinity sites, the best mean-square deviation between observed and calculated values, D, depends on the choice of (average) drug binding constant to carrier DNA, Kd.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA sequence preferences of several AT-selective minor groove binding ligands.

We have examined the interaction of distamycin, netropsin, Hoechst 33258 and berenil, which are AT-selective minor groove-binding ligands, with synthetic DNA fragments containing different arrangements of AT base pairs by DNase I footprinting. For fragments which contain multiple blocks of (A/T)4 quantitative DNase I footprinting reveals that AATT and AAAA are much better binding sites than TTAA and TATA. Hoechst 33258 shows that greatest discrimination between these sites with a 50-fold difference in affinity between AATT and TATA. Alone amongst these ligands, Hoechst 33258 binds to AATT better than AAAA. These differences in binding to the various AT-tracts are interpreted in terms of variations in DNA minor groove width and suggest that TpA steps within an AT-tract decrease the affinity of these ligands. The behaviour of each site also depends on the flanking sequences; adjacent pyrimidine-purine steps cause a decrease in affinity. The precise ranking order for the various binding sites is not the same for each ligand.     

Methidiumpropyl-EDTA.Fe(II) and DNase I footprinting report different small molecule binding site sizes on DNA.

DNase I and MPE.Fe (II) footprinting both employ partial cleavage of ligand-protected DNA restriction fragments and Maxam-Gilbert sequencing gel methods of analysis. One method utilizes the enzyme, DNase I, as the DNA cleaving agent while the other employs the synthetic molecule, methidium-propyl-EDTA (MPE). For actinomycin D, chromomycin A3 and distamycin A, DNase I footprinting reports larger binding site sizes than MPE.Fe (II). DNase I footprinting appears more sensitive for weakly bound sites. MPE.Fe (II) footprinting appears more accurate in determining the actual size and location of the binding sites for small molecules on DNA, especially in cases where several small molecules are closely spaced on the DNA. MPE.Fe (II) and DNase I report the same sequence and binding site size for lac repressor protein on operator DNA.     

The 0 degree C closed complexes between Escherichia coli RNA polymerase and two promoters, T7-A3 and lacUV5

The promoter-specific binding of Escherichia coli RNA polymerase to the T7-A3 and the lacUV5 promoters at 0 degrees C was analyzed by DNase I footprinting. At 37 degrees C, the footprint from RNA polymerase bound to the A3 promoter is essentially the same as that reported by Galas, D.J., and Schmitz, A., (1978) Nucleic Acids Res. 5, 3157-3170 for the lacUV5 promoter. At 0 degrees C, the footprint for the A3 promoter is well defined but reduced in size. The principal difference between the 0 and 37 degrees C footprints is a region from -2 to +18 which is protected by polymerase at the higher but not at the lower temperature. In contrast, the 0 degree C footprint for the lacUV5 promoter differs substantially in character from the footprint for A3 at 0 degree C. The footprint is similar to the pattern of DNase I digestion of DNA bound to a surface; alternating regions of sensitive and protected DNA are spaced at intervals of about 10 base pairs. This region of DNase I-sensitive and -resistant DNA has the same boundaries as the 0 degree C footprint on T7-A3. Temperature shift experiments confirmed the sequence specificity of the RNA polymerase interaction with UV5 at 0 degree C. These results indicate that RNA polymerase binds specifically to each promoter sequence in a closed complex. The increased time and amounts of RNA polymerase required to form the 0 degree C footprint on the lacUV5 promoter indicate that it binds RNA polymerase more weakly than does the T7-A3 promoter. Therefore there is a correlation between the binding constant for closed complex formation estimated from kinetic measurements and the formation of the 0 degree C footprint. The -35 region of the promoter may be more important in establishing the 0 degree C footprint because the T7-A3 promoter is a better match to the consensus sequence. Conversely, the -10 region seems less important because lacUV5 is a perfect match to the consensus, whereas the T7-A3 promoter matches at only five out of seven positions. The 0 degree C footprints encompass both regions along with the spacer; the combination of these regions rather than an individual region may determine the character of the footprint and the magnitude of the binding constant.     

Analysis of chromosome III replicators reveals an unusual structure for the ARS318 silencer origin and a conserved WTW sequence within the origin recognition complex binding site

Saccharomyces cerevisiae chromosome III encodes 11 autonomously replicating sequence (ARS) elements that function as chromosomal replicators. The essential 11-bp ARS consensus sequence (ACS) that binds the origin recognition complex (ORC) has been experimentally defined for most of these replicators but not for ARS318 (HMR-I), which is one of the HMR silencers. In this study, we performed a comprehensive linker scan analysis of ARS318. Unexpectedly, this replicator depends on a 9/11-bp match to the ACS that positions the ORC binding site only 6 bp away from an Abf1p binding site. Although a largely inactive replicator on the chromosome, ARS318 becomes active if the nearby HMR-E silencer is deleted. We also performed a multiple sequence alignment of confirmed replicators on chromosomes III, VI, and VII. This analysis revealed a highly conserved WTW motif 17 to 19 bp from the ACS that is functionally important and is apparent in the 228 phylogenetically conserved ARS elements among the six sensu stricto Saccharomyces species.     

Topography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication.

Investigation of the DNA binding properties of the simian virus 40 (SV40) A protein (large T antigen) and the hybrid adenovirus-SV40 D2 protein revealed that both viral proteins protect similar regions of SV40 DNA from digestion by DNase I or methylation by dimethyl sulfate. However, the interaction of D2 protein with DNA was more sensitive to increases of NaCl concentration than was the interaction of wild-type SV40 A protein. Dimethylsulfate footprinting identified 13 DNA pentanucleotide contact sites at the viral origin of replication. The sequences of these sites corresponded to the consensus family 5'-(G greater than T) (A greater than G)GGC-3'. The pentanucleotides were distributed in three regions of origin DNA. Region I contained three pentanucleotide contact sites arranged as direct repetitions encompassing a span of 23 base pairs. In region II, four pentanucleotides were oriented as inverted repetitions that also spanned a total of 23 base pairs. Region III had six recognition pentanucleotides arranged as direct repetitions in a space of 59 base pairs. These fundamental variations in DNA arrangement are likely to determine different patterns of protein binding in each region.     

Identification of the sequences required for chromosomal replicator function in Kluyveromyces lactis

The analysis of replication intermediates of a Kluyveromyces lactis chromosomal autonomous replicating sequence (ARS), KARS101, has shown that it is active as a chromosomal replicator. KARS101 contains a 50 bp sequence conserved in two other K. lactis ARS elements. The deletion of the conserved sequence in KARS101 completely abolished replicator activity, in both the plasmids and the chromosome. Gel shift assays indicated that this sequence binds proteins present in K. lactis nuclear extracts, and a 40 bp sequence, previously defined as the core essential for K. lactis ARS function, is required for efficient binding. Reminiscent of the origin replication complex (ORC), the binding appears to be ATP dependent. A similar pattern of protection of the core was seen with in vitro footprinting. KARS101 also functions as an ARS sequence in Saccharomyces cerevisiae. A comparative study using S. cerevisiae nuclear extracts revealed that the sequence required for binding is a dodecanucleotide related to the S. cerevisiae ARS consensus sequence and essential for S. cerevisiae ARS activity.