Site-dependent cleavage of pBR322 DNA by restriction endonuclease HinfI.

Cleavage of pBR322 DNA I by the restriction endonuclease HinfI is preferentially inhibited at specific HinfI cleavage sites. These sites in pBR322 DNA I have been identified and ordered with respect to the frequency with which they are cleaved. The HinfI site most resistant to cleavage in pBR322 DNA I is unique in that runs of G-C base pairs are immediately adjacent on both sites. Two differently permuted linear (DNA III) species were produced by cleavage with two different restriction endonucleases, PstI and AvaI. Only one of these linear molecules, that produced by PstI, exhibits the same preferential cleavage pattern as DNA I. The second linear species, that arising from AvaI digestion, shows pronounced relative inhibition of cleavage at the HinfI sites nearest the ends of the molecule (100 to 120 base pairs away, respectively). This result suggest that proximity to the termini of a linear DNA molecule might also influence preferential cleavage. The possibility of formation of stem-loop structures does not appear to influence preferential cleavage by HinfI.     

The effect of differential methylation by Escherichia coli of plasmid DNA and phage T7 and lambda DNA on the cleavage by restriction endonuclease MboI from Moraxella bovis.

The nucleotide sequence recognized and cleaved by the restriction endonuclease MboI is 5' GATC and is identical to the central tetranucleotide of the restriction sites of BamHI and BglII. Experiments on the restriction of DNA from Escherichia coli dam and dam+ confirm the notion that GATC sequences are adenosyl-methylated by the dam function of E. coli and thereby are made refractory to cleavage by MboI. On the basis of this observation the degree of dam methylation of various DNAs was examined by cleavage with MboI and other restriction endonucleases. In plasmid DNA essentially all of the GATC sequences are methylated by the dam function. The DNA of phage lambda is only partially methylated, extended methylation is observed in the DNA of a substitution mutant of lambda, lambda gal8bio256, and in the lambda derived plasmid, lambdadv93, which is completely methylated. In contrast, phage T7 DNA is not methylated by dam. A suppression of dam methylation of T7 DNA appears to act only in cis dam. A suppression of dam methylation of T7 DNA appears to act only in cis since plasmid DNA replicated in a T7-infected cell is completely methylated. The results are discussed with respect to the participation of the dam methylase in different replication systems.     

Location in bacteriophage lamdba DNA of cleavage sites of the site-specific endonuclease from Bacillus amyloliquefaciens H.

The sites in Escherichia coli bacteriophage lambda DNA cleaved by the site-specific endonuclease isolated from Bacillus amyloliquefaciens H (BamI) are found to be at 0.114, 0.466, 0.580, 0.713, and 0.861 lambda units. The sites were located by analysis of the products of digestion of lambda DNA and lambda-ara transducing phage DNA, and verified by double digestion with BamI and EcoRI.     

Bacillus subtilis RNAase III cleavage sites in phage SP82 early mRNA

We have determined the DNA sequence encoding three sites in Bacillus subtilis phage SP82 early mRNA that are cleaved by a B. subtilis processing endonuclease. The products generated by cleavage of the RNA were sequenced to determine the exact points of RNA strand scission. We propose that the RNA surrounding each processing site forms a stable stem-loop structure and that cleavage occurs at the 5- side of specific adenosine residues located on the loop. The model is consistent with our previous observations that the active site of the enzyme recognizes double-stranded RNA. S1 mapping experiments with RNA-DNA hybrids established that the same cleavage sites are used both in vivo and in vitro. Examination of the B. subtilis processing sites on SP82 mRNA reveals distinctive features of primary and secondary structure that are not present in any of the E. coli RNAase III processing sites previously studied.     

Specificity and mode of cleavage of the pH 4.0 endonuclease from adenovirus type 2 - infected KB cells.

Adenovirus type 2 or lambda DNA was digested with the pH 4.0 endonuclease, purified from adenovirus 2-infected KB cells. The enzyme produces a limit digest of approximate size in the range of 140-210 base pairs long. The termini of the DNA fragments generated by the endonuclease digestion had 3'-P and 5'-OH groups. The 3' and 5' end groups of the products were analyzed. Our data indicate that 3' end group was a purine (68-76%), dA occuring about twice the frequency of dG. The 5' end group was either dG or dC with equal frequency. Data obtained by treatment of the 5' labeled endonuclease product of lambda DNA with single-strand specific S1 nuclease from Asperigillus oryzae or exonuclease VII from Escherichia coli indicated that the majority of the products had a short 5' protruding ends. The mode of cleavage of this endonuclease seems to be through initial formation of several single-strand breaks with some base specificity. If these breaks are at close proximity on opposite strands, double-stranded fragments with protruding ends are generated.     

Association between DNA cleavage during apoptosis and regions of chromatin replication

We have addressed the association between the site of DNA cleavage during apoptosis and DNA replication. DNA double strand breaks were introduced into chromatin containing pulse labeled nascent DNA by the induction of apoptosis or autocleavage of isolated nuclei. The location of these breaks in relation to nascent DNA were revealed by Bal 31 exonuclease digestion at the cut sites. Our data show that Bal31 accessible cut sites are directly linked to regions enriched in nascent DNA. We suggest that these regions coincide with the termini of replication domains, possibly linked by strong DNA-matrix interactions with biophysically defined topological structures of 0.5 - 1.3 Mbp in size. The 50 kbp fragments that are commonly observed as products of apoptosis are also enriched in nascent DNA within internal regions but not at their termini. It is proposed that these fragments contain a subset of replicon DNA that is excised during apoptosis through recognition of their weak attachment to the nuclear matrix within the replication domain.J. Cell. Biochem. 70:604-615, 1998. © 1998 Wiley-Liss, Inc. © 1998 Wiley-Liss, Inc.     

The DNA restriction endonuclease of Escherichia coli B. II. Further studies of the structure of DNA intermediates and products

The DNA intermediates and final products formed by the Type I restriction endonuclease, EcoB, were further characterized. DNA cleaved on only one strand (hemi-restricted DNA) contains gaps of approximately 70-100 nucleotides, while the fully restricted products contain 3'-single-stranded tails averaging approximately 70-100 nucleotides for each strand cleaved. The gaps and tails are formed with the release of an equal number of nucleotides as small oligonucleotides that are soluble in acid. After purification, neither the hemi-restricted nor the fully restricted DNAs are cleaved again by EcoB. There is no apparent specificity for which strand of a duplex is initially cleaved by EcoB, nor is there specificity with respect to the composition of the 3'-terminal nucleotide formed on the DNA or the 3'- or 5'-terminal nucleotides of the acid-soluble oligonucleotides released during DNA cleavage. The structure formed at the 5' terminus of the DNA product which blocks phosphorylation by T4 polynucleotide kinase remains unknown, but its removal with phage lambda exonuclease allows at least some reutilization of recognition sites by EcoB as well as phosphorylation of the newly formed 5' termini. To explain the complex mechanism of this enzyme, it is suggested that the unidentified 5'-tails prevent wasteful rerestriction from occurring, whereas the 3'-single-stranded tails create DNA which, when nonhomologous to chromosomal DNA, cannot be rescued because such tails are not substrate for DNA polymerases. However, when homologous chromosomal DNA exists, the randomly cleaved large fragments with these tails can easily be assimilated by recA-mediated genetic recombination, thus stimulating DNA exchange between related organisms.     

Electron microscopic heteroduplex study and restriction endonuclease cleavage analysis of the DNA genomes of three lactic streptococcal bacteriophages

Three lactic streptococcal bacteriophages were compared with one another by electron microscopic analysis of heteroduplex DNA molecules. The phages were almost identical in morphology and had been isolated over a period of 10 years on different strains of Streptococcus cremoris from cheese plants situated in different parts of New Zealand. There was a high degree of homology between the DNAs, in agreement with Southern blot hybridization data reported earlier. There were, however, distinct regions of nonhomology, mostly between 0.45 and 1.71 kilobases in length, suggestive of the occurrence of block recombination events. A deletion of 2.23 kilobases in the two more recently isolated phages, or an insertion in the first isolate, was found. All three phage DNAs showed differences in restriction endonuclease cleavage sites. Alignment of the restriction endonuclease maps with the heteroduplex maps showed that differences in cleavage sites occurred most frequently in regions of nonhomology. However, differences in cleavage sites in regions of apparent homology were also detected, indicating that point mutations may have occurred in addition to block recombination events.     

Electron microscopic heteroduplex study and restriction endonuclease cleavage analysis of the DNA genomes of three lactic streptococcal bacteriophages.

Three lactic streptococcal bacteriophages were compared with one another by electron microscopic analysis of heteroduplex DNA molecules. The phages were almost identical in morphology and had been isolated over a period of 10 years on different strains of Streptococcus cremoris from cheese plants situated in different parts of New Zealand. There was a high degree of homology between the DNAs, in agreement with Southern blot hybridization data reported earlier. There were, however, distinct regions of nonhomology, mostly between 0.45 and 1.71 kilobases in length, suggestive of the occurrence of block recombination events. A deletion of 2.23 kilobases in the two more recently isolated phages, or an insertion in the first isolate, was found. All three phage DNAs showed differences in restriction endonuclease cleavage sites. Alignment of the restriction endonuclease maps with the heteroduplex maps showed that differences in cleavage sites occurred most frequently in regions of nonhomology. However, differences in cleavage sites in regions of apparent homology were also detected, indicating that point mutations may have occurred in addition to block recombination events.     

Effect of in vitro cleavage of apurinic/apyrimidinic sites on bleomycin-induced mutagenesis of repackaged lambda phage

Previous studies have revealed bleomycin to be a potent base-substitution mutagen in repackaged phage lambda. In order to assess the role of apurinic/apyrimidinic (AP) sites in bleomycin-induced mutagenesis, bleomycin-damaged lambda DNA was treated with putrescine or endonuclease IV to effect cleavage of bleomycin-induced AP sites. The DNA was then packaged, the phage grown in SOS-induced E. coli, and the frequency of clear-plaque mutants in the progeny was determined. Bleomycin-induced mutagenesis was decreased approx. 2-fold by treating the DNA with putrescine, but was unaffected by endonuclease IV. The results are consistent with the production of bleomycin-induced mutation at certain AP sites having a closely opposed single-strand break, since such sites are cleaved by putrescine but not by endonuclease IV.     

An endonuclease activity of venom phosphodiesterase specific for single-stranded and superhelical DNA.

A homogeneous preparation of venom phosphodiesterase from Crotalus adamanteus possesses an intrinsic endonuclease activity, specific for superhelical (form I) and single-stranded DNA. The phosphodiesterase degrades single-stranded T7 DNA by endonucleolytic cleavages. Duplex T7 DNA is hydrolyzed by the liberation of acid-soluble products simultaneously from the 3' and 5' termini but without demonstrable internal scissions in duplex regions. Since venom phosphodiesterase is known to hydrolyze oligonucleotides stepwise from the 3' termini, the cleavage at the 5' end of duplex T7 DNA is ascribed to an endonuclease activity. Form I PM2 DNA is nicked to yield first relaxed circles and then linear DNA which is subsequently hydrolyzed only from the chain termini. The linear duplex DNA intermediates consist of a discrete series of fragments (11 are usually resolved on agarose gels) with initial molecular weights ranging from 6.3 x 10(6) (the intact PM2 DNA size) to approximately 1 x 10(6). The cleavage of the form I molecule must, therefore, occur at a limited number of unique sites. The enzyme also cleaves nonsuperhelical, covalently closed circular PM2 DNA but at a 10(4) times slower rate. Both the endonuclease activity on form I DNA and the known exonuclease activity co-migrate on polyacrtkanude gels, are optimally active at pH 9, are stimulated by small concentrations of Mg2+, and are similarly inactivated by heat, reducing agents, and EDTA.     

Effect of in vitro cleavage of apurinic/apyrimidinic sites on bleomycin-induced mutagenesis of repackaged lambda phage.

Previous studies have revealed bleomycin to be a potent base-substitution mutagen in repackaged phage lambda. In order to assess the role of apurinic/apyrimidinic (AP) sites in bleomycin-induced mutagenesis, bleomycin-damaged lambda DNA was treated with putrescine or endonuclease IV to effect cleavage of bleomycin-induced AP sites. The DNA was then packaged, the phage grown in SOS-induced E. coli, and the frequency of clear-plaque mutants in the progeny was determined. Bleomycin-induced mutagenesis was decreased approx. 2-fold by treating the DNA with putrescine, but was unaffected by endonuclease IV. The results are consistent with the production of bleomycin-induced mutation at certain AP sites having a closely opposed single-strand break, since such sites are cleaved by putrescine but not by endonuclease IV.     

Correlated genetic and EcoRI cleavage map of Bacillus subtilis bacteriophage phi105 DNA.

The seven previously identified EcoRI cleavage fragments of phi 105 DNA were ordered with respect to their sites of origin on the phage genome by marker rescue. One fragment, H, did not carry any determinants essential for replication. This fragment was totally missing in a deletion mutant which exhibited a lysogenization-defective phenotype. There is a nonessential region on the phi 105 genome which begins in fragment B, spans fragment H, and ends in fragment F. The size of the nonessential region, as estimated by alterations observed in the fragmentation patterns of deletion mutant DNAs, is approximately 2.7 X 10(6) daltons. Two new EcoRI cleavage fragments with molecular weights of approximately 0.2 X 10(6) were detected by autoradiography of 32P-labeled DNA. These small fragments were not located on the cleavage map.     

A fluorimetric assay for on-line detection of DNA cleavage by restriction endonucleases

We have developed an assay for online detection of DNA cleavage by restriction endonucleases, suitable for the high throughput screening of the activity and flanking sequence preference of restriction endonuclease variants. For this purpose oligodeoxynucleotides were used, labeled with either 6-FAM or TAMRA whose fluorescence is quenched by a neighboring DABCYL group. After endonucleolytic cleavage the products are too short to remain double-stranded and the fluorophor labeled strand is released with concomitant increase in fluorescence which can be easily quantified. Employing this method, cleavage reactions can be monitored continuously, allowing for fast detection of specific activity as well as determination of kinetic parameters. To demonstrate the reliability of our assay we measured K(M) and k(cat) values for the restriction endonuclease EcoRV and obtained results similar to those obtained with established assays. Moreover, our method makes it possible to observe the cleavage of two different substrates differing in the sequences flanking the EcoRV site and labeled with different fluorophors in competition in a single experiment. This assay can be carried out in a microplate format, which allows for the analysis of many restriction endonuclease variants in parallel.     

Studies of viable T4 bacteriophage containing cytosine-substituted DNA (T4dC phage)

Summary Digestion of non-glucosylated and cytosine-substituted T4 phage (T4dC) DNA with SalI restriction endonuclease showed that the DNA had nine SalI-sensitive sites. There were eight SalI sites in DNA from a strain which had a deletion in the rII-denB-ndd region. The comparison of two digestion patterns indicated that one of the SalI-sensitive sites was present in the deleted region and that the SalI-F fragment (8.4x10⁶ daltons) was located adjacent to the SalI-C or SalI-D fragment (15.5x10⁶ daltons) on the T4 chromosome. The DNA gave no detectable cleavage product when digested with BamHI endonuclease alone, while, when digested successively with BamHI and SalI, the DNA yielded two new digestion products in place of one fragment formed by SalI alone. The BamHI-sensitive site was in the SalI-A fragment (25.2x10⁶ daltons). The usefulness of this information for making cleavage maps of T4 phage chromosome is discussed.     

Kinetic studies on the cleavage of adenovirus DNA by restriction endonuclease Eco RI.

The kinetics of cleavage of DNA from Adenovirus Type 1 (Ad1), Type 5 (Ad5) and Type 6 (Ad6) by restriction endonuclease EcoRI was investigated by quantitative evaluation of the fluorescence from ethidium stained DNA fragments separated on agarose gels. The apparent rate constants of cleavage at different cleavage sites have been determined and large differences in the cleavage rates of the individual sites within one type of DNA were found. From the kinetics of cleavage information on the sequence of the DNA fragments can be obtained. The order of the fragment A, B, C, D of Ad6 DNA obtained after complete cleavage by restriction endonuclease Eco RI was found to be A-D-C-B; the order of the corresponding fragments A, B, C of Ad1 and Ad5 DNA was found to be A-C-B.     

Primary and secondary structure specificity of the cleavage of 'single-stranded' DNA by endonuclease Hinf I.

The interaction of endonuclease Hinf I with single-stranded fd DNA was examined. The sizes of the cleavage products indicate that the enzyme cuts this substrate at the same sequences as double-stranded DNA (GANTC). To determine whether or not the recognition sites in single-stranded DNA have to be present in double-stranded form in order to be cleaved, DNA fragments containing complementary or non-complementary Hinf I sequences were prepared and treated as substrates. The results suggest that completely base-paired recognition sites are necessary for cleavage. Sequences surrounding the Hinf I pentanucleotides significantly modulate the reaction rates.     

In vitro maturation of circular bacteriophage P2 DNA. Purification of ter components and characterization of the reaction

The protein components required for generation of cohesive ends in vitro from circular bacteriophage P2 DNA have been purified to near homogeneity. In the presence of ATP, the purified products of P2 genes M and P together with empty phage capsids (comprised primarily of the N protein) mediate site-specific cleavage of circular P2 DNA at the cohesive end site (cos). This terminase or ter system also utilizes circular DNAs of bacteriophages P4 and 186, introducing site-specific scissions at cos sites within these molecules. The ter reaction exhibits a peculiar requirement for a circular DNA substrate. Substrate activity is greatly reduced when circular P2, P4, or 186 DNAs are linearized by restriction endonuclease hydrolysis. Furthermore, multimeric P4 DNA molecule sites are also essentially inactive in the linear form but are active in the circular state. The dependence of ter action on a circular substrate is not due to inhibition of the system by linear DNA, nor does it appear to reflect a requirement for substrate superhelicity since circular P4 DNA containing single strand scissions is subject to terminase action. The terminase reaction is supported by ATP, dATP, or beta, gamma-imido ATP, but not by other ribonucleoside triphosphates ADP, alpha, beta-methylene ATP, or beta, gamma-methylene ATP. A DNA-dependent ATPase, which hydrolyzes ATP to AMP, copurifies with the P2 P protein and is inactivated with the same kinetics as P activity upon treatment with N-ethylmaleimide. The ATPase does not display specificity for P2 DNA in vitro.