DNAase I,DNAase II and staphylococcal nuclease cut at different,yet symmetrically located,sites in the nucleosome core

We have determined the relative location of pancreatic DNAase (DNAase I), spleen acid DNAase (DNAase II) and staphylococcal nuclease cleavage sites in the nucleosome core. Each of these three enzymes cleaves the DNA of chromatin at 10. n nucleotide intervals (n integer); this specificity presumably reflects the internal structure of the nucleosome. We have already reported that DNAase I cleaves nucleosomal DNA so that nearest adjacent cuts on opposite strands are staggered by 2 nucleotides, 3′ end extending (Sollner-Webb and Felsenfeld, 1977). Here we show that the nearest cuts made by DNAase II in nucleosomal DNA are staggered by 4 nucleotides, 3′ end extending, while cuts made by staphylococcal nuclease have a stagger of 2 nucleotides, 5′ end extending. The cutting sites of the three enzymes thus do not coincide. Each pair of staggered cuts, however, is symmetrically located about a common axis-that is, the “dyad axes” that bisect nearest pairs of cutting sites coincide for all three enzymes. This result is consistent with the presence of a true dyad axis in the nucleosome core.Our results support the conclusion that a structural feature of the nucleosome, having a 10 nucleotide periodicity, is the common recognition site for all three nucleases. The position of the cut is determined, however, by the individual characteristics of each enzyme. Sites potentially available to nuclease cleavage span a region of 4 nucleotides out of this 10 nucleotide repeat, and a large fraction of these sites are actually cut. Thus much of the nucleosomal DNA must in some sense be accessible to the environment.     

Restriction endonuclease BpuJI specific for the 5'-CCCGT sequence is related to the archaeal Holliday junction resolvase family

Type IIS restriction endonucleases (REases) recognize asymmetric DNA sequences and cleave both DNA strands at fixed positions downstream of the recognition site. REase BpuJI recognizes the asymmetric sequence 5′-CCCGT, however it cuts at multiple sites in the vicinity of the target sequence. We show that BpuJI is a dimer, which has two DNA binding surfaces and displays optimal catalytic activity when bound to two recognition sites. BpuJI is cleaved by chymotrypsin into an N-terminal domain (NTD), which lacks catalytic activity but binds specifically to the recognition sequence as a monomer, and a C-terminal domain (CTD), which forms a dimer with non-specific nuclease activity. Fold recognition approach reveals that the CTD of BpuJI is structurally related to archaeal Holliday junction resolvases (AHJR). We demonstrate that the isolated catalytic CTD of BpuJI possesses end-directed nuclease activity and preferentially cuts 3nt from the 3′-terminus of blunt-ended DNA. The nuclease activity of the CTD is repressed in the apo-enzyme and becomes activated upon specific DNA binding by the NTDs. This leads to a complicated pattern of specific DNA cleavage in the vicinity of the target site. Bioinformatics analysis identifies the AHJR-like domain in the putative Type III enzymes and functionally uncharacterized proteins.     

Packaging of coliphage lambda DNA. I. The role of the cohesive end site and the gene A protein

The cohesive ends of the DNA of bacteriophage λ particles are normally formed by the action of a nuclease on the cohesive end sites (cos) of concatemeric λ DNA (reviewed by Hohn et al., 1977). The nuclease also cuts the cos site of an integrated prophage, and DNA located to the right is preferentially packaged into phage particles. This process occurs with approximately the same efficiency and rate in a single lysogen as in a tandem polylysogen. Thus, the rate of cos cutting does not increase when the number of cos sites per molecule increases, an hypothesis that has been proposed to explain why cohesive ends are not formed in circular monomers of λ DNA. We propose instead that the interaction of Ter with cos is influenced by the configuration of the DNA outside of cos during packaging, and that this configuration is different for circular monomers than for other forms of λ DNA. A model that gives rise to such a difference is described.We also found that missense mutations in the λ A gene changed the efficiency of packaging of phage relative to host DNA. This was not the case for missense mutations in several phage genes required for capsid formation. Thus, the product of gene A plays a role in determining packaging specificity, as expected if it is or is part of the nuclease that cuts λ DNA at cos.     

Subunit assembly and mode of DNA cleavage of the type III restriction endonucleases EcoP1I and EcoP15I

DNA cleavage by type III restriction endonucleases requires two inversely oriented asymmetric recognition sequences and results from ATP-dependent DNA translocation and collision of two enzyme molecules. Here, we characterized the structure and mode of action of the related EcoP1I and EcoP15I enzymes. Analytical ultracentrifugation and gel quantification revealed a common Res(2)Mod(2) subunit stoichiometry. Single alanine substitutions in the putative nuclease active site of ResP1 and ResP15 abolished DNA but not ATP hydrolysis, whilst a substitution in helicase motif VI abolished both activities. Positively supercoiled DNA substrates containing a pair of inversely oriented recognition sites were cleaved inefficiently, whereas the corresponding relaxed and negatively supercoiled substrates were cleaved efficiently, suggesting that DNA overtwisting impedes the convergence of the translocating enzymes. EcoP1I and EcoP15I could co-operate in DNA cleavage on circular substrate containing several EcoP1I sites inversely oriented to a single EcoP15I site; cleavage occurred predominantly at the EcoP15I site. EcoP15I alone showed nicking activity on these molecules, cutting exclusively the top DNA strand at its recognition site. This activity was dependent on enzyme concentration and local DNA sequence. The EcoP1I nuclease mutant greatly stimulated the EcoP15I nicking activity, while the EcoP1I motif VI mutant did not. Moreover, combining an EcoP15I nuclease mutant with wild-type EcoP1I resulted in cutting the bottom DNA strand at the EcoP15I site. These data suggest that double-strand breaks result from top strand cleavage by a Res subunit proximal to the site of cleavage, whilst bottom strand cleavage is catalysed by a Res subunit supplied in trans by the distal endonuclease in the collision complex.     

Multiple sequence-specific DNA binding activities are eluted from chicken nuclei at low ionic strengths.

DNA sequence-specific binding proteins eluted from chicken erythrocyte and thymus nuclei, and fractionated as described by Emerson and Felsenfeld (19), have been investigated by filter binding and footprint analyses. The erythrocyte nuclear protein fraction specifically binds to at least two sites within the 5' flanking chromatin hypersensitive site of the chicken beta A-globin gene, and to a site 5' to the human beta-globin gene. The major chicken beta A globin gene binding site [G)18CGGGTGG) and the human beta-globin gene binding site [TA)6(T)8C(T)4) occur at or near sequences which are hypersensitive to S1 nuclease cleavage in supercoiled plasmids. Downstream, the second chicken beta A-globin gene binding site includes the beta-globin gene CACCC consensus sequence. Filter binding studies also show other sequence specific binding activities to human N-ras and human (but not chicken) c-myc gene sequences.     

Nucleosome arrangement in alpha-satellite chromatin of African green monkey cells.

By analyzing the accessibility of restriction endonuclease sites in African green monkey alpha-satellite chromatin, we demonstrate the absence of a unique phase relationship between nucleosomes and alpha-satellite DNA. The data indicate a minimum of three different positions for nucleosome cores relative to the alpha-satellite sequence and suggest a random distribution in at least some regions. In addition, while we confirm published reports that staphylococcal nuclease cuts the alpha-satellite sequence in chromatin at a highly preferred site, two-dimensional gel electrophoresis of nuclear digests demonstrates that this site is preferentially cut by staphylococcal nuclease even when it is within the nucleosome core. These data indicate that staphylococcal nuclease is not useful for determining nucleosome positions on alpha-satellite DNA, and perhaps on other specific DNA sequences as well.     

Specific cleavage of DNA molecules at RecA-mediated triple-strand structure

A novel procedure to cleave DNA molecules at any desired base sequence is presented. This procedure is based upon our finding that double-stranded DNA molecules at a site where RecA-mediated triple-stranded DNA structure with a complimentary deoxyoligonucleotide is located can be cleaved by a single-strand specific nuclease, such as nuclease S1 or BAL31, between the first base at the 5′ termini of the deoxyoligonucleotides and the nearest base proximal to the 5′ termini. Accordingly, the sequence as well as the number of the cleavage sites to be cleaved can be custom designed by selecting deoxyoligonucleotides with specific base sequences for triple-stranded DNA formation. The basic characteristics of the cleavage reaction and typical applications of the procedure are presented with actual results, including those which involve cleavage of complex genomic DNA at the very sites one desires.     

The use of micrococcal nuclease as a probe for drug-binding sites on DNA

The cutting pattern produced by micrococcal nuclease on three DNA fragments has been determined in the absence and presence of various DNA-binding drugs. The enzyme itself cuts almost exclusively at pA and pT bonds, showing a greater activity at (A-T)n than in homopolymeric runs of A and T. Each drug produces distinct changes in the cleavage pattern. The protected regions can not be pinpointed with sufficient precision to assess the exact drug-binding sites on account of the sequence selectivity of the enzyme, although where a direct comparison is possible these include most of those seen as DNAase I footprints. The enzyme is most useful for assessing the selectivity of drugs which bind to AT-rich regions. Several drugs protect the DNA from micrococcal nuclease attack in regions which do not contain their acknowledged best binding sites. It appears that micrococcal nuclease is sensitive to the existence of secondary drug-binding sites which are not evident with other footprinting techniques.     

C5 cytosine methylation at CpG sites enhances sequence selectivity of mitomycin C-DNA bonding

We have established that UvrABC nuclease is equally efficient in cutting mitomycin C (MC)-DNA monoadducts formed at different sequences and that the degree of UvrABC cutting represents the extent of drug-DNA bonding. Using this method we determined the effect of C5 cytosine methylation on the DNA monoalkylation by MC and the related analogues N-methyl-7-methoxyaziridinomitosene (MS-NMA) and 10-decarbamoylmitomycin C (DC-MC). We have found that C5 cytosine methylation at CpG sites greatly enhances MC and MS-NMA DNA adduct formation at those sites while reducing adduct formation at non-CpG sequences. In contrast, although DC-MC DNA bonding at CpG sites is greatly enhanced by CpG methylation, its bonding at non-CpG sequences is not appreciably affected. These cumulative results suggest that C5 cytosine methylation at CpG sites enhances sequence selectivity of drug-DNA bonding. We propose that the methylation pattern and status (hypo- or hypermethylation) of genomic DNA may determine the cells' susceptibility to MC and its analogues, and these effects may, in turn, play a crucial role in the antitumor activities of the drugs.     

Control region of SV40 minichromosomes is preferentially cleaved by single-strand specific S1 nuclease

We have analysed S1 sensitivity of SV40 minichromosomes isolated from the nuclei of infected cells at the late stage of infection. We show that a fraction of purified minichromosomes is sensitive towards double-strand cleavage by S1 nuclease. The pattern of specific cleavage reminiscent of that found for subcloned fragment under supercoiling is superimposed upon apparently random double-strand cuts along the entire regulatory region. Therefore, the cleavage sites are not exclusively confined to the regions with the reported alternate DNA conformation.     

Pancreatic DNAase cleavage sites in nuclei

The DNA of nuclei is cleaved by a variety of nucleases in such a way that the cuts on a given strand are always separated by an integral multiple of 10 nucleotides. However, the spacing between cutting sites on opposite strands is not known for any nuclease. In this paper, we describe the determination of the spacing, or stagger, between cuts on opposite strands produced by the action of pancreatic DNAase (DNAase I) on nuclei. When nuclei are digested with DNAase I and the resultant DNA is analyzed by gel electrophoresis without prior denaturation, a complex pattern of bands is observed. A method which gives better than 90% recovery of DNA from polyacrylamide gels was used to isolate the individual fractions corresponding to these bands. The structure of the fractions was then determined using single-strand-specific nuclease to digest single-stranded "tails" and using DNA polymerases to extend recessed 3'-OH termini of partially duplex regions. Our results show that each component consists of a double-stranded region terminating in single-stranded tails at both ends. Although both chains of every duplex are 10-n nucleotides long (n integer), the chains are never completely paired. The experiments with DNA polymerase show an abundance of structures in which the 3'-OH termini of these duplexes are recessed by 8 nucleotides, and by inference, there must be structures with 5'-P termini recessed by 2 or 12 nucleotides. Thus DNAase I acts on nuclei to produce DNA with staggered cuts on opposite strands, separated by (10-n + 8) and (10-n + 2) base pairs (with 5'-P and 3'-OH termini extending, respectively). Two classes of models of DNA folding in the nucleosome have been proposed by other investigators to account for the presence of DNAase I cleavage sites at 10-n intervals along each DNA chain. One class of models leads to the prediction that cuts should either be unstaggered or separated by 10 nucleotides, while the other class is consistent with staggers of 6 and 4 nucleotides. Neither prediction is verified by our data; however, all these models may be made consistent with the results by assuming that the enzyme's site of recognition on nucleosomal DNA is not the same as its site of cleavage.     

A specific 3'' exonuclease activity of UvrABC.

Specific cutting of undamaged DNA by UvrABC nuclease is observed. It occurs seven nucleotides (nt) from the 3' terminus of oligonucleotides annealed to single-stranded M13 DNA circles. Although the location of the UvrABC cut on undamaged DNA is similar to that of the cut on the 5' side of a damaged DNA site during the dual incision reaction, the cut of undamaged DNA is not an intermediate in the dual incision step. On DNA duplexes with a single AAF adduct, the anticipated cut at the eighth phosphodiester bond 5' of the lesion is present, but extra cuts at 7-nt increments are observed at the 15th and 22nd phosphodiester bonds. We suggest that these additional cuts are made by the UvrABC activity observed on undamaged DNA; such activity is referred to as ABC 3' exonuclease and may play a significant role by providing a suitable gap for RecA-mediated recombinational exchanges during repair of interstrand crosslinks and closely opposed lesions. This ABC 3' exonuclease activity depends on higher concentrations of Uvr proteins as compared with dual incision and may be relevant to reactions that occur when UvrA and UvrB are increased during SOS induction.     

Mung bean nuclease exhibits a generalized gene-excision activity upon purified Plasmodium falciparum genomic DNA.

A novel set of reaction conditions for mung bean nuclease has been described in which Plasmodium genes were specifically excised as intact fragments from purified DNA. We have now determined that under the new conditions mung bean nuclease cleaves precisely at sites outside of the coding region of every P. falciparum gene for which the extent of the protein coding region in genomic DNA is known. We conclude that this enzyme activity is probably a general one for P. falciparum genes. Introns are not specifically cleaved, although one gene contained a cleavage site within an intron. There is no direct relationship between dA.dT-richness and sites of cleavage under these conditions. Also contrary to the expectations of a model based on cleavage at denaturation bubbles, there was no general relationship between the concentration of the DNA denaturant, formamide, and the size of the resulting gene-containing fragments. Thus, the data strongly suggest the involvement of an altered DNA structure near gene boundaries in determining the recognition sites for this enzyme activity.     

Staphylococcal nuclease makes a single non-random cut in the simian virus 40 viral minichromosome.

When compact simian virus 40 (SV40) minichromosomes are treated with staphylococcal nuclease at 0 °C under limit-digest conditions, about one-third of the minichromosomes remain resistant to nuclease, a third of them are nicked, while the remaining third suffer one and only one double-stranded cut. Results show that each cleaved minichromosome is cut only once and afterwards becomes resistant to further fragmentation. This is in marked contrast to the action of staphylococcal nuclease at 37 °C, which leads to a rapid fragmentation of all minichromosomes to oligo- and mononucleosomes.The SV40 linear DNA III produced by low-temperature nuclease digestion of minichromosomes was redigested with single-cut restriction endonucleases. By this mapping procedure it was determined that the location of the staphylococcal nuclease cut is neither unique nor random; it occurs at a number of discrete sites on the DNA, half of all cuts being concentrated at the origin of replication and nearby in the “late” portion of the SV40 genome. Control experiments have shown that when staphylococcal nuclease digests naked SV40 DNA at 0 °C it does not “hesitate” after the first cut. Although initial cuts in the purified DNA are non-random in location, their distribution is quite different from that generated by a low-temperature nuclease digestion of compact SV40 minichromosomes. Possible interpretations of these results are discussed in view of the recent finding that a specific region of the SV40 genome is uniquely exposed in the minichromosome (Varshavsky et al., 1978, 1979; Scott &; Wigmore, 1978).     

Hypersensitive mung bean nuclease cleavage sites in Plasmodium knowlesi DNA

Nucleotide sequences of Plasmodium knowlesi DNA that are cleaved by mung bean nuclease (Mbn) at low enzyme concentration (0.2 units enzyme per micrograms DNA) are listed. They are tandemly repeated purine/pyrimidine (RpY) stretches of DNA with (ApT) dimers predominating. Most cut sites are within almost 100% RpY tracts. The enzyme cleaves at many points within the RpY stretch and usually hydrolyzes the 5'-ApT-3' linkage. These alternating RpY target sites are flanked by homopurine and homopyrimidine stretches. At least one Mbn target site lies next to an in vivo transcribed region.     

Control Region of SV40 Minichromosomes is Preferentially Cleaved by Single-Strand Specific SI Nuclease

Abstract

We have analysed SI sensitivity of SV40 minichromosomes isolated from the nuclei of infected cells at the late stage of infection. We show that a fraction of purified minichromosomes is sensitive towards double-strand cleavage by SI nuclease. The pattern of specific cleavage reminiscent ofthat found for subcloned fragment under supercoiling is superimposed upon apparently random double-strand cuts along the entire regulatory region. Therefore, the cleavage sites are not exclusively confined to the regions with the reported alternate DNA conformation.