Interaction of Escherichia coli primase with a phage G4ori(c)-E. coli SSB complex.

We earlier reported that Escherichia coli single-stranded DNA-binding protein (SSB) bound in a fixed position to the stem-loop structure of the origin of complementary DNA strand synthesis in phage G4 (G4ori(c)), leaving stem-loop I and the adjacent 5' CTG 3', the primer RNA initiation site, as an SSB-free region (W. Sun and G. N. Godson, J. Biol. Chem. 268:8026-8039, 1993). Using a small 278-nucleotide (nt) G4ori(c) single-stranded DNA fragment that supported primer RNA synthesis, we now demonstrate by gel shift that E. coli primase can stably interact with the SSB-G4ori(c) complex. This stable interaction requires Mg2+ for specificity. At 8 mM Mg2+, primase binds to an SSB-coated 278-nt G4ori(c) fragment but not to an SSB-coated control 285-nt LacZ ss-DNA fragment. In the absence of Mg2+, primase binds to both SSB-coated fragments and gives a gel shift. T4 gene 32 protein cannot substitute for E. coli SSB in this reaction. Stable interaction of primase with naked G4ori(c). single-stranded DNA was not observed. DNase I and micrococcal nuclease footprinting, of both 5' and 3' 32P-labeled DNA, demonstrated that primase interacts with two regions of G4ori(c): one covering stem-loop I and the 3' sequence flanking stem-loop I which contains the pRNA initiation site and another located on the 5' sequence flanking stem-loop III.     

Nucleosomes and subnucleosomes: heterogeneity and composition

Previous studies (Varshavsky, Bakayev and Georgiev, 1976a) have shown that chromatin subunits (mononucleosomes) and their oligomers in a mild staphylococcal nuclease digest of chromatin display a heterogeneous content of histone H1. We now report that a mild staphylococcal nuclease digest of either chromatin or nuclei from mouse Ehrlich tumor cells contains mononucleosomes of three discrete kinds. The smallest mononucleosome (MN₁) contains all histones except H1 and a DNA fragment 140 base pairs (bp) long. The intermediate mononucleosome (MN₂) contains all five histones and a DNA fragment 170 bp long. The third mononucleosome (MN₃) also contains all five histones, but its DNA fragment is longer and more heterogeneous in size (180–200 bp). Most of the MN₃ particles are rapidly converted by nuclease into mononucleosomes MN₁ and MN₂ There exists, however, a relatively nuclease-resistant subpopulation of the MN₃ mononucleosomes. These 200 bp MN₁ particles contain not only histones but also nonhistone proteins, and are significantly more resistant to nuclease than the bulk of MN₃ particles and the smaller mononucleosomes MN₁ and MN₂.There are eight major kinds of staphylococcal nuclease-produced soluble subnucleosomes (SN). The SN₁ is a set of naked double-stranded DNA fragments ～20 bp long. The SN₂ is a complex of a specific basic nonhistone protein (molecular weight ～16,000 daltons) and a DNA fragment ～27 bp long. The SN₃ contains histone H4, the above-mentioned specific nonhistone protein and a DNA fragment ～27 bp long. The SN₄ contains histones H2a, H2b, H4 and a DNA fragment ～45 bp long. The SN₅ contains histones H2a, H2b, H3 and a DNA fragment ～55 bp long. The SN₆ is a complex of histone H1 and a DNA fragment ～35 bp long. Subnucleosomes SN₇ and SN₈ each contain all the histones except H1, and DNA fragments ～100 and ～120 bp long, respectively.Nuclease digestion of isolated mono- or dinucleosomes does not produce some of the subnucleosomes. These and related findings indicate that the cleavage required to generate these subnucleosomes result from some aspect of chromatin structure which is lost upon digestion to mono- and dinucleosomes.     

Sequence-specific interaction of histones with the simian virus 40 enhancer region in vitro

DNA fragments containing either one or both of the 72-base pair (bp) elements which constitute the SV40 enhancer and the three adjacent 21-bp repeats were associated with histone octomers from chicken erythrocytes in vitro. Both fragments formed complexes with electrophoretic mobilities of nucleosomes containing the appropriate length of DNA. Analysis of DNase I cutting of uniquely end-labeled complexes suggests that the fragment containing a single 72-bp element forms a positioned core particle. Control experiments show that positioning is not due to the 21-bp repeats or to end effects. The fragment with a tandem repeat of the 72-bp element also does not associate randomly with histones. The data are consistent with formation of a core particle on one or the other of the repeated enhancer sequences. We discuss possible functional consequences of such nucleosome positioning.     

Gadd45, a p53-responsive stress protein, modifies DNA accessibility on damaged chromatin

This report demonstrates that Gadd45, a p53-responsive stress protein, can facilitate topoisomerase relaxing and cleavage activity in the presence of core histones. A correlation between reduced expression of Gadd45 and increased resistance to topoisomerase I and topoisomerase II inhibitors in a variety of human cell lines was also found. Gadd45 could potentially mediate this effect by destabilizing histone-DNA interactions since it was found to interact directly with the four core histones. To evaluate this possibility, we investigated the effect of Gadd45 on preassembled mononucleosomes. Our data indicate that Gadd45 directly associates with mononucleosomes that have been altered by histone acetylation or UV radiation. This interaction resulted in increased DNase I accessibility on hyperacetylated mononucleosomes and substantial reduction of T4 endonuclease V accessibility to cyclobutane pyrimidine dimers on UV-irradiated mononucleosomes but not on naked DNA. Both histone acetylation and UV radiation are thought to destabilize the nucleosomal structure. Hence, these results imply that Gadd45 can recognize an altered chromatin state and modulate DNA accessibility to cellular proteins.     

Purification and characterization of the Ner repressor of bacteriophage Mu

The Ner protein of bacteriophage Mu acts as a lambda cro-like negative regulator of the phage's early (transposase) operon. Using the band retardation assay to monitor ner-operator-specific DNA-binding activity, the 8 kDa Ner protein was purified to homogeneity. DNase I footprinting revealed that the purified protein bound and protected a specific DNA operator that contains two 12 bp sites with the consensus sequence 5'-ANPyTAPuCTAAGT-3', separated by a 6 bp spacer region. Moreover, regions corresponding to a turn of the DNA helix flanking these 12 bp repeats are also protected by Ner. Unlike the functionally similar lambda cro protein, gel filtration experiments show that the native molecular mass of Mu Ner to be approx. 8 kDa. These results, plus the pattern of DNase I protection, suggest that the protein may bind as a monomer to each of its specific DNA substrates.     

DNA protection by histone-like protein HU from the hyperthermophilic eubacterium Thermotoga maritima

In mesophilic prokaryotes, the DNA-binding protein HU participates in nucleoid organization as well as in regulation of DNA-dependent processes. Little is known about nucleoid organization in thermophilic eubacteria. We show here that HU from the hyperthermophilic eubacterium Thermotoga maritima HU bends DNA and constrains negative DNA supercoils in the presence of topoisomerase I. However, while binding to a single site occludes approximately 35 bp, association of T. maritima HU with DNA of sufficient length to accommodate multiple protomers results in an apparent shorter occluded site size. Such complexes consist of ordered arrays of protomers, as revealed by the periodicity of DNase I cleavage. Association of TmHU with plasmid DNA yields a complex that is remarkably resistant to DNase I-mediated degradation. TmHU is the only member of this protein family capable of occluding a 35 bp nonspecific site in duplex DNA; we propose that this property allows TmHU to form exceedingly stable associations in which DNA flanking the kinks is sandwiched between adjacent proteins. We suggest that T. maritima HU serves an architectural function when associating with a single 35 bp site, but generates a very stable and compact aggregate at higher protein concentrations that organizes and protects the genomic DNA.     

Weak strand displacement activity enables human DNA polymerase beta to expand CAG/CTG triplet repeats at strand breaks

Using synthetic DNA constructs in vitro, we find that human DNA polymerase beta effectively catalyzes CAG/CTG triplet repeat expansions by slippage initiated at nicks or 1-base gaps within short (14 triplet) repeat tracts in DNA duplexes under physiological conditions. In the same constructs, Escherichia coli DNA polymerase I Klenow Fragment exo(-) is much less effective in expanding repeats, because its much stronger strand displacement activity inhibits slippage by enabling rapid extension through two downstream repeats into flanking non-repeat sequence. Polymerase beta expansions of CAG/CTG repeats, observed over a 32-min period at rates of approximately 1 triplet added per min, reveal significant effects of break type (nick versus gap), strand composition (CTG versus CAG), and dNTP substrate concentration, on repeat expansions at strand breaks. At physiological substrate concentrations (1-10 microm of each dNTP), polymerase beta expands triplet repeats with the help of weak strand displacement limited to the two downstream triplet repeats in our constructs. Such weak strand displacement activity in DNA repair at strand breaks may enable short tracts of repeats to be converted into longer, increasingly mutable ones associated with neurological diseases.     

Periodicity and fragment size of DNA from mouse TLT hepatoma chromatin and chromatin fractions using endogenous and exogenous nucleases

Summary The action of micrococcal nuclease, DNase I and DNase II on mouse TLT hepatoma chromatin revealing the periodicity of its structure as visualized by denaturing and nondenaturing gel electrophoresis, was consistent with the action of these enzymes on other chromatins. Micrococcal nuclease showed a complex subnucleosome fragment pattern based on multiples of 10 base pairs with a prominant couplet at 140/160 base pairs and the absence of the 80 base pair fragment. This couplet of the core and minimal nucleosome fragments was conspicuously present in the mononucleosomes found in the 11S fractions of a glycerol gradient centrifugation. DNase I and II produced a fairly even distribution of a 10 base pair increasing series of fragments to about 180 base pairs, a pattern also repeated in the DNA of nucleosome glycerol-gradient fractions. In limited digestions by these nucleases multinucleosomic DNA fragments are pronounced. These fragment lengths are multiples of an estimated average repeat length of nucleosome DNA of 180 base pairs. The action of the endogenous Mg/Ca-stimulated endonuclease produced only limited cuts in the hepatoma chromatin resulting primarily in multi-nucleosommc DNA fragment lengths and only upon lengthy digestion limited subnucleosomic, 10-base-pair multiple fragments are produced. The putative euchromatin-enriched fractions (50–75S) of the glycerol gradient centrifugation of autodigested chromatin, similarly, contained primarily the multinucleosomic DNA fragment lengths. These results are consistent with our previous electron microscopic demonstration that autodigested chromatin as well as the putative euchromatin-enriched fractions were composed of multinucleosomic chromatin segments containing a full complement of histones.     

Quantitation of DNase I sensitivity in Xenopus chromatin containing active and inactive globin, albumin and vitellogenin genes.

The disappearance of defined restriction fragments of the beta 1-globin, an albumin and the A1 vitellogenin gene was quantitated after DNase I digestion and expressed by a sensitivity factor defined by a mathematical model. Analysis of naked DNA showed that the gene fragments have similar but not identical sensitivity factors. DNase I digestion of chromatin revealed for the same gene fragments sensitivity factors differing over a much wilder range. This is correlated to the activity of the genes analyzed: the beta 1-globin gene fragment is more sensitive to DNase I in chromatin of erythrocytes compared to hepatocytes whereas the albumin gene fragment is more sensitive to DNase I in chromatin of hepatocytes. The A1 vitellogenin gene has the same DNase I sensitivity in both cell types. Comparing the DNase I sensitivity of the three genes in their inactive state we suggest that different chromatin conformations may exist for inactive genes.     

The gamma delta resolvase bends the res site into a recombinogenic complex.

We have characterized complexes between the gamma delta resolvase and its recombination site, res, using both a gel retardation assay and DNase I cleavage. The mobility of resolvase-res complexes in polyacrylamide gels is sensitive to the location of res within the DNA fragment and is at a minimum when res is at its center. This behavior is characteristic of a protein-dependent bend. By the same assay we have found that bends are induced upon the binding of resolvase to each of the three individual binding sites that constitute res. In the wild-type res, the centers of binding sites I and II are 53 bp apart and the central section of the intersite DNA is sensitive to DNase I cleavage. We find that insertions of 10 or 21 bp (one or two turns of the DNA helix) have no discernible effect on the ability of res to recombine or to form complexes with resolvase. However, insertions of short segment (e.g. 6 or 17 bp) equivalent to nonintegral numbers of helical turns, inhibit recombination and prevent the formation of the normally compact resolvase-res complex. Complexes of resolvase with res containing 10 or 21 bp insertions exhibit a pattern of enhanced and suppressed DNase I cleavages that suggest that the intersite segment is curved. This curvature requires both that site I and II are appropriately spaced, and that site III is also present and occupied.     

Structural features of the integration site of foreign DNA in the transgenic mouse genome

The structure of the transgenic mouse DNA region containing an integrated transgene (fragment of pBR322 sequence) was analysed. In one of the sequences flanking the transgene, short direct and inverted overlapping repeats were revealed at a distance of 60 bp from the integration site. In the same flanking sequence, there is an extended sequence (3.5 kbp) 0.3-1 kbp away from the transgene. It repeats 100-300 times in the mouse genome and is highly conservative (the homologs of the repeat have been revealed in other mammalian, bird, fish and insect genomes). This up-to-date unknown family of highly-conserved dispersed repeats has been denoted by T1. We believe that both the revealed short inverted repeats capable of forming hairpins with loops and the T1 repeat are structures involved in the process of non-homologous insertion of foreign DNA into the region of the transgenic mouse genome.     

Chromatin remodeling facilitates DNA incision in UV-damaged nucleosomes

The DNA repair machinery must locate and repair DNA damage all over the genome. As nucleosomes inhibit DNA repair in vitro, it has been suggested that chromatin remodeling might be required for efficient repair in vivo. To investigate a possible contribution of nucleosome dynamics and chromatin remodeling to the repair of UV-photoproducts in nucleosomes, we examined the effect of a chromatin remodeling complex on the repair of UV-lesions by Micrococcus luteus UV endonuclease (ML-UV endo) and T4-endonuclease V (T4-endoV) in reconstituted mononucleosomes positioned at one end of a 175-bp long DNA fragment. Repair by ML-UV endo and T4-endoV was inefficient in mononucleosomes compared with naked DNA. However, the human nucleosome remodeling complex, hSWI/SNF, promoted more homogeneous repair by ML-UV endo and T4-endo V in reconstituted nucleosomes. This result suggests that recognition of DNA damage could be facilitated by a fluid state of the chromatin resulting from chromatin remodeling activities.     

DNA binding specificity of the bovine papillomavirus E1 protein is determined by sequences contained within an 18-base-pair inverted repeat element at the origin of replication.

Bovine papillomavirus type 1 (BPV-1) DNA replicates episomally and requires two virally expressed proteins, E1 and E2, for this process. Both proteins bind to the BPV-1 genome in the region that functions as the origin of replication. The binding sequences for the E2 protein have been characterized previously, but little is known about critical sequence requirements for E1 binding. Using a bacterially expressed E1 fusion protein, we examined binding of the BPV-1 E1 protein to the origin region. E1 strongly protected a 28-bp segment of the origin (nucleotides 7932 to 15) from both DNase I and exonuclease III digestion. Additional exonuclease III protection was observed beyond the core region on both the 5' and 3' sides, suggesting that E1 interacted with more distal sequences as well. Within the 28-bp protected core, there were two overlapping imperfect inverted repeats (IR), one of 27 bp and one of 18 bp. We show that sequences within the smaller, 18-bp IR element were sufficient for specific recognition of DNA by E1 and that additional BPV-1 sequences beyond the 18-bp IR element did not significantly increase origin binding by E1 protein. While the 18-bp IR element contained sequences sufficient for specific binding by E1, E1 did not form a stable complex with just the isolated 18-bp element. Formation of a detectable E1-DNA complex required that the 18-bp IR be flanked by additional DNA sequences. Furthermore, binding of E1 to DNA containing the 18-bp IR increased as a function of overall increasing fragment length. We conclude that E1-DNA interactions outside the boundaries of the 18-bp IR are important for thermodynamic stabilization of the E1-DNA complex. However, since the flanking sequences need not be derived from BPV-1, these distal E1-DNA interactions are not sequence specific. Comparison of the 18-bp IR from BPV-1 with the corresponding region from other papillomaviruses revealed a symmetric conserved consensus sequence, T-RY--TTAA--RY-A, that may reflect the specific nucleotides critical for E1-DNA recognition.     

Affinity of HMG17 for a mononucleosome is not influenced by the presence of ubiquitin-H2A semihistone but strongly depends on DNA fragment size.

We have used a two-dimensional (deoxyribonucleoprotein leads to DNA) electrophoretic binding assay to study the interaction of the purified high mobility group protein HMG17 with isolated HeLa mononucleosomes as a function of their DNA fragment size and the presence of ubiquitin-H2A semihistone. No significant differences between affinities of HMG17 for ubiquitinated and non-ubiquitinated core mononucleosomes were observed. In striking contrast, the apparent affinity of HMG17 for a mononucleosome increases more than 100-fold upon an increase of the length of the mononucleosomal DNA fragment by as few as 3 to 5 bp over the core DNA length (integral of 146 bp). We suggest that the magnitude of this effect is sufficient to explain the preferential binding of HMG17 in vitro to mononucleosomes derived from actively transcribed genes.     

DNase I footprint of ABC excinuclease

The incision and excision steps of nucleotide excision repair in Escherichia coli are mediated by ABC excinuclease, a multisubunit enzyme composed of three proteins, UvrA, UvrB, and UvrC. To determine the DNA contact sites and the binding affinity of ABC excinuclease for damaged DNA, it is necessary to engineer a DNA fragment uniquely modified at one nucleotide. We have recently reported the construction of a 40 base pair (bp) DNA fragment containing a psoralen adduct at a central TpA sequence (Van Houten, B., Gamper, H., Hearst, J. E., and Sancar, A. (1986a) J. Biol. Chem. 261, 14135-14141). Using similar methodology a 137-bp fragment containing a psoralen-thymine adduct was synthesized, and this substrate was used in DNase I-footprinting experiments with the subunits of ABC excinuclease. It was found that the UvrA subunit binds specifically to the psoralen modified 137-bp fragment with an apparent equilibrium constant of K8 = 0.7 - 1.5 X 10(8) M-1, while protecting a 33-bp region surrounding the DNA adduct. The equilibrium constant for the nonspecific binding of UvrA was Kns = 0.7 - 2.9 X 10(5) M-1 (bp). In the presence of the UvrB subunit, the binding affinity of UvrA for the damaged substrate increased to K8 = 1.2 - 6.7 X 10(8) M-1 while the footprint shrunk to 19 bp. In addition the binding of the UvrA and UvrB subunits to the damaged substrate caused the 11th phosphodiester bond 5' to the psoralen-modified thymine to become hypersensitive to DNase I cleavage. These observations provide evidence of an alteration in the DNA conformation which occurs during the formation of the ternary UvrA.UvrB.DNA complex. The addition of the UvrC subunit to the UvrA.UvrB.DNA complex resulted in incisions on both sides of the adduct but did not cause any detectable change in the footprint. Experiments with shorter psoralen-modified DNA fragments (20-40 bp) indicated that ABC excinuclease is capable of incising a DNA fragment extending either 3 or 1 bp beyond the normal 5' or 3' incision sites, respectively. These results suggest that the DNA beyond the incision sites, while contributing to ABC excinuclease-DNA complex formation, is not essential for cleavage to occur.     

Avian Retrovirus pp32 DNA-Binding Protein I. Recognition of Specific Sequences on Retrovirus DNA Terminal Repeats

The avian retrovirus pp32 protein possesses a DNA-nicking activity which prefers supercoiled DNA as substrate. We have investigated the binding of pp32 to avian retrovirus long terminal repeat (LTR) DNA present in both supercoiled and linear forms. The cloned viral DNA was derived from unintegrated Schmidt-Ruppin A (SRA) DNA. A subclone of the viral DNA in pBR322 (termed pPvuII-DG) contains some src sequences, tandem copies of LTR sequences, and partial gag sequences in the order src-U(3) U(5):U(3) U(5)-gag. Binding of pp32 to supercoiled pPvuII-DG DNA followed by digestion of this complex with a multicut restriction enzyme (28 fragments total) permitted pp32 to preferentially retain on nitrocellulose filters two viral DNA fragments containing only LTR DNA sequences. In addition, pp32 also preferentially retained four plasmid DNA fragments containing either potential promoters or Tn3 "left-end" inverted repeat sequences. Mapping of the pp32 binding sites on viral LTR DNA was accomplished by using the DNase I footprinting technique. The pp32 protein, but not the avian retrovirus alphabeta DNA polymerase, is able to form a unique protein-DNA complex with selected regions of either SRA or Prague A LTR DNAs. Partial DNase I digestion of a 275-base pair SRA DNA fragment complexed with pp32 gives upon electrophoresis in denaturing gels a unique ladder pattern, with regions of diminished DNase I susceptibility from 6 to 10 nucleotides in length, in comparison with control digests in the absence of protein. The binding of pp32 to this fragment also yields enhanced DNase I-susceptible sites that are spaced between the areas protected from DNase I digestion. The protected region of this unique complex was a stretch of 170 +/- 10 nucleotides that encompasses the presumed viral promoter site in U(3), which is adjacent to the src region, extends through U(5), and proceeds past the joint into U(3) for about 34 base pairs. No specific protection or DNase I enhancement by pp32 was observed in experiments with a 435-base pair SRA DNA fragment derived from a part of U(3) and the adjacent src region or a 55-base pair DNA fragment derived from another part of U(3). The DNA sequence of Prague A DNA at the fused LTRs differs from that of SRA DNA. The alteration in the sequence at the juncture of the LTRs prevented pp32 from forming a stable complex in this region of the LTR. Our results are relevant to two aspects of the interaction between pp32 and LTR DNA. First, the pp32 protein in the presence of selected viral DNA restriction fragments possibly forms a higher order oligomer analogous to Escherichia coli DNA gyrase-DNA complexes or eucaryotic nucleosome structures. Second, the specificity of the binding suggests a role for pp32 and the protected DNA sequences in the retrovirus life cycle. The preferred sequences to which pp32 binds include two adjacent 15-base pair inverted terminal repeats at the joint between U(5) and U(3) in SRA DNA. This region is involved in circularization of linear DNA and is perhaps the site that directs integration into cellular DNA.     

Slipped (CTG).(CAG) repeats of the myotonic dystrophy locus: surface probing with anti-DNA antibodies

At least 15 human diseases have been associated with the length-dependent expansion of gene-specific (CTG).(CAG) repeats, including myotonic dystrophy (DM1) and spinocerebellar ataxia type 1 (SCA1). Repeat expansion is likely to involve unusual DNA structures. We have structurally characterized such DNA, with (CTG)(n).(CAG)(n) repeats of varying length (n=17-79), by high-resolution gel electrophoresis, and have probed their surfaces with anti-DNA antibodies of known specificities. We prepared homoduplex S-DNAs, which are (CTG)x.(CAG)y where x=y, and heteroduplex SI-DNAs, which are hybrids where x>y or x相似文献