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Restoration by T4 ligase of DNA sequences sensitive to 'flush' cleaving restriction enzyme
Abstract
Fourteen “flush”-ended segments originate from the action of the restriction endonuclease Hae III of Haemophilus aegiptius
on the DNA of the colicinogenic factor ColE 1 (A. Oka and M. Takanami, Nature, 264, 191, 1976). They are joined by the T4
polynucleotide ligase. The reaction can be monitored by gel electrophoresis, electron microscopy and resistance to phosphatase
of the 5'- 32P labelled ends. The joined products are a random recombination of the original segments, and can be cleaved by the same Hae
III endonuclease to restore the exact electrophoretic pattern of the Hae III-cut ColE 1 DNA.
In a properly diluted mixture of 5'-32P segments treated with T4 ligase, the level of phosphatase resistance is very close to the frequency of circle-formation
as determined by electron microscopy: thus, the joining of the “flush”-ends involves the formation of circular structures
covalently closed in both strands.
The study of human mutagenesis requires methods of measuring somatic mutations in normal human tissues and inherited mutations in human populations. Such methods should permit measurement of rare mutations in the presence of abundant wild-type copies and should be general to the human genome. A sensitivity of 2 x 10-6 for point mutations was recently achieved in human cells using a novel method of target isolation, constant denaturant capillary electrophoresis (CDCE), and high-fidelity polymerase chain reaction (hifi-PCR) (Li-Sucholeiki and Thilly, 2000). This method is applicable to 100-base pair (bp) DNA domains juxtaposed with a naturally occurring domain of a higher melting temperature, or a natural clamp. Such sequence domains represent about 9% of the human genome. To permit analysis of rare point mutations in the human genome more generally, this thesis developed a procedure in which a clamp can be ligated to any 100-bp sequence of interest. This procedure was combined with the previous method to create a new method of point mutational analysis that is not dependent on a naturally occurring clamp. To demonstrate the new method, a sequence with a natural clamp, a part of the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene (cDNA-bp 223-318), was analyzed using both the natural and ligated clamps. A sensitivity of 2 x 10-5 in human cells was demonstrated using the ligated clamp as opposed to 5 x 10-6 using the natural clamp.
The study of human mutagenesis requires methods of measuring somatic mutations in normal human tissues and inherited mutations in human populations. Such methods should permit measurement of rare mutations in the presence of abundant wild-type copies and should be general to the human genome. A sensitivity of 2 x 10-6 for point mutations was recently achieved in human cells using a novel method of target isolation, constant denaturant capillary electrophoresis (CDCE), and high-fidelity polymerase chain reaction (hifi-PCR) (Li-Sucholeiki and Thilly, 2000). This method is applicable to 100-base pair (bp) DNA domains juxtaposed with a naturally occurring domain of a higher melting temperature, or a natural clamp. Such sequence domains represent about 9% of the human genome. To permit analysis of rare point mutations in the human genome more generally, this thesis developed a procedure in which a clamp can be ligated to any 100-bp sequence of interest. This procedure was combined with the previous method to create a new method of point mutational analysis that is not dependent on a naturally occurring clamp. To demonstrate the new method, a sequence with a natural clamp, a part of the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene (cDNA-bp 223-318), was analyzed using both the natural and ligated clamps. A sensitivity of 2 x 10-5 in human cells was demonstrated using the ligated clamp as opposed to 5 x 10-6 using the natural clamp. (Cont.) The sensitivity of the new method using the ligated clamp was demonstrated to be limited by the fidelity of Pfu DNA polymerase used for PCR. The sequence of the ligated clamp accounted for the differences in sensitivity as a result of causing a decreased efficiency of mutant enrichment by CDCE. The new method can be applied to measure somatic mutations in normal human tissues, such as lung tissues, in which point mutations at fractions above 10-5 have been observed. This method can also detect predominant inherited mutations even for genes carrying recessive deleterious alleles in pooled samples derived from a large number of individuals. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2002. Includes bibliographical references (leaves 176-200).
This chapter discusses deoxyribonucleic acid (DNA)-joining enzymes. The first DNA-joining enzymes identified were DNA ligases; they join together DNA chains by transmuting the high-energy pyrophosphate linkage of a nucleotide cofactor into a phosphoester bond between the 5′-phosphoryl and 3′-hydroxyl termini. DNA ligase from Escherichia coli (E. coli) is a single polypeptide chain of a molecular weight of 74,000, and bacteriophage T4 induces a DNA ligase having a single chain with a molecular weight of 68,000. Phosphodiester bond synthesis is coupled to the cleavage of a pyrophosphate bond in nicotinamide adenine dinucleotide (NAD) for the bacterial enzyme and in adenosine triphosphate (ATP) for the phage enzyme. An E. coli ligase joins oligo(dT) that is base paired to poly(dA); it also joins oligo(dA) base paired to poly(dT), but this is a much less favorable reaction. The self-complementary poly[d(A-T)] can fold back upon itself to oppose the ends, and a ligase seals these into closed loop molecules (circles). T4 DNA ligase is a much more permissive enzyme than an E. coli ligase; it catalyzes a number of ligations that cannot be performed with the E. coli enzyme.
As part of our investigations on the relationship between DNA structure and gene regulation, a 352-base pair Hae III fragment was cloned containing the leftward operator-promoter (PL) region of bacteriophage lambda. This was accomplished without the aid of a phenotypic assay for the cloned fragment. A Hae III digest of a segment of the lambda genome was first fractionated by RPC-5 column chromatography. The partially purified PL fragment was then ligated into the Eco RI site of the pBR322 plasmid vector and cloned into the recBC+ Escherichia coli host C600(R-M-) using a technique that converts the Hae III ends of the fragment into Eco RI sites. Similar cloning attempts into a recBC- host (C600-SF8) were unsuccessful. The cloned fragment has the PL promoter oriented toward the tetracycline resistance genes of the vector, and is isolated from the plasmid (pRW601) by digestion with Eco RI followed by sucrose gradient sedimentation. The fragment was identified as PL by restriction mapping, repressor binding, sequencing, and promoter location. The now complete sequence of this fragment, part of which was known previously, reveals a large A/T-rich region immediately adjacent to the PL promoter. We have generated deletions in this region in order to study the influence of this sequence on promoter function.
The temperature dependence of the T4 DNA ligase-catalyzed joining of plasmid DNA linearized by the action of HaeIII, EcoRI
and PstI restriction endonucleases has been investigated by electron microscopy analysis. The extent of joining is maximal at 4° and decreases with increasing temperatures following sigmoid - like curves.
The temperature at which 50% of the maximal reaction is still observable increases going from DNA termini without single-stranded
overlaps (produced by HaeIII) to termini with four nucleotides overlap, composed only by two A and two T (produced by EcoRI) to termini with four nucleotide overlap, composed by A, T, G and C (produced by PstI)
Plasmids are widely used as vectors and methods of their isolation can vary depending upon the host organism. On the basis of phenotypic effect and organization in the chromosomes, genes can be subdivided into various groups, such as simple genes, complex genes, operons, regulons, and multiple regulons. Any DNA segments obtained by shear—by using two different restriction enzymes or by using restriction enzymes that produce blunt ends—can be joined without adding homopolymer tails. However, blunt-end ligation does not allow the recovery of the cloned fragment after the recombinant DNA has been propagated. The restriction site DNA linkers used for manipulating DNAs that lack cohesive ends (sheared DNA, synthetic DNA, cDNA)—are commercially available. If the DNA fragment is not flush-ended, it can be first digested with the Aspergillus nuclease S1 to produce even ends as this linker can be used with several restriction enzymes. Individual competent cells can be transformed by more than one plasmid simultaneously, which allows for the indirect selection of a cryptic plasmid if the transformation is carried out with a mixture of a cryptic plasmid and a small selectable plasmid.
A restriction-like enzyme has been purified from Haemophilus aegyptius. This nuclease, endonuclease Z, produces a rapid decrease in the viscosity of native calf thymus and H. influenzae deoxyribonucleic acids (DNA), but does not degrade homologous DNA. The specificity of endonuclease Z is different from that of the similar endonuclease isolated from H. influenzae (endonuclease R). The purified enzyme cleaves the double-stranded replicative form DNA of bacteriophage phiX174 (phiX174 RF DNA) into at least 11 specific limit fragments whose molecular sizes have been estimated by gel electrophoresis. The position of these fragments with respect to the genetic map of phiX174 can be determined by using the genetic assay for small fragments of phiX174 DNA.
The polynucleotide ligase isolated from T4-infected Escherichia coli was previously shown to bring about repair of breaks in the single strands of bihelical DNA. The present work shows that the enzyme can also catalyze the joining of DNA duplexes at their base-paired ends. This novel reaction occurs when the deoxynucleoside at a 5'-end carries a phosphate group and the complementary deoxynucleoside opposite to it carries a 3'-hydroxyl group. The consequence is the lengthening of the original duplex to form dimers or oligomers depending upon whether one or both ends are base-paired.
A method for the isolation of T4-amber-N82-induced DNA ligase is described which results in a nearly homogeneous enzyme preparation after two column chromatographic steps. The enzyme is detected during the purification by its ability to form a stable acid-precipitable enzyme-adenylate complex. Some properties of the assay, such as the effect of salt, temperature and incubation time, are presented. The isolated enzyme and its adenylate complex are characterized by acrylamide gel electrophoresis under native and denaturing conditions, as well as by isoelectric focusing. The purified enzyme exhibits a molecular weight of approximately 60000. Isoelectric focusing yields at least 5 protein components, which are able to form an enzyme-adenylate complex. The main activity possesses a pI of 6. The enzyme preparation is capable of repairing T5+ DNA known to contain about 4 or 5 single-strand breaks, to circularize λ DNA and to join HindIII and EcoRI fragments.
An electron microscopic examination of λ DNA, partially denatured at high pH, shows that the sections which denature first are at the same positions as those previously observed for λ DNA partially denatured by heat.A similar study of BU†-labeled λ DNA shows the A·BU-rich regions to be situated at similar positions along the molecule to the A·T-rich regions in normal λ DNA, but it also shows that the A·BU-rich regions melt out at a lower pH.Details of a modification to the protein film technique are given in the Materials and Methods section.
Double-stranded DNA fragments terminated at their 5′-ends by the singlestranded sequence pA-A-T-T-, generated by digestion of DNA with EcoRI restriction endonuclease, were ligated with Escherichia coli polynucleotide ligase under various conditions of temperature, concentration and time. The linear and circular products of ligation were separated by electrophoresis in agarose gel and quantitated by densitometry. The rate of ligation of (EcoRI-cleaved) simian virus (SV40) DNA at a concentration of 100 μg/ml increased from 0 °C to 5 °C to 10 °C (6-fold increase overall); raising the temperature to 15 °C did not further increase the rate of ligation. At the appropriate DNA concentrations, the predominant products of ligation are either linear concatemers that are integral multimers of the starting DNA fragment, or covalently closed circular structures of the monomeric DNA fragment. Ligating a mixture of two different length DNA fragments gives rise to all of the possible expected recombinant molecules.Linear or circular products of ligation were predicted by consideration of the total concentration of DNA termini, i, and the local concentration of one terminus in the neighborhood of the other on the same DNA molecule, j. The parameter j is a function of the length of a DNA molecule, providing this length is greater than the random coil segment of DNA. Experimentally it was found that circular structures are formed in significant amounts only under conditions when the value of j is several times greater than that of i. When j = i, equal amounts of linear and circular products would be expected, but most of the molecules were ligated into linear concatemers. No circular structure of a DNA fragment whose contour length l (6 × 10−2 μm) is smaller than the random coil segment value b (7·17 × 10−2 μm) was observed, while circular structures of the dimer of the same molecule (12 × 10−2 μm) were detected.
COLICIN E1 plasmid (colE1) is a closed circular DNA molecule with a molecular weight of 4.2 × 106 (ref. 1). ColE1 DNA has extensively been used as a molecular vehicle for cloning and amplification of DNA in genetic engineering2,3. In order to expand such investigations, it is useful to make a cleavage map ordering colEl DNA pieces produced by bacterial restriction endonucleases. Escherichia coli RI restriction endonuclease (R . EcoRI) has been shown to cleave colE1 DNA at a single unique site4-6. We have now constructed a physical map with the EcoRI site as a reference point using two restriction endonucleases from Haemophilus aegyptius (R . HaeII and R . HaeIII). The map of the colE1 moiety involved in a hybrid plasmid pML21 (ref. 7) carrying about a half of the colEl genome was also determined. Restriction endonucleases and fragments produced by the cleavages were abbreviated as those by Smith and Nathans8. The size of each fragment was expressed as a percentage of the length of the colEl DNA molecule.
By a triester chemical synthesis method, three decameric DNA's have been made; these act as substrates for several restriction
endonucleases, including Eco RI, Bam I, and Hind III. These homogenous decamers form duplexes that can be efficiently blunt-end
ligated to themselves or to other DNA molecules by the action of T4 DNA ligase and thus are useful tools for molecular cloning
experiments.
Linear double-stranded molecules of the circularly permuted and terminally redundant DNA of Salmonella bacteriophage P22 have been converted to oligomeric products in the presence of polynucleotide ligase coded for by the coliphage T4. The reaction has been monitored by sucrose density-gradient centrifugation and electron microscopy. It goes slowly and gives yields of 30-40%. The products are mainly dimers and trimers, but higher oligomers are also present.
DNA ligase extracted from uninfected Escherichia coli seems unable to perform a similar reaction, which is concluded to involve the fully base-paired termini.
Linear double-stranded molecules of simian virus(SV) 40 DNA, produced by the action of the bacterial restriction endonuclease R1, are oligomerized by either ligase; therefore, this reaction seems to involve single-stranded cohesive ends. No mixed products could be found when P22 DNA and linear SV 40 DNA were exposed together to the T4 ligase.
Electrophoresis on slab gels containing a linear gradient of polyacrylamide concentration has been used to separate DNA fragments obtained by restriction of viral DNAs. A simple method of preparing gradient gels using a sucrose density-gradient mixer and preexisting slab gel apparatus is described. DNA fragments of molecular weights 7 × 104–14 × 106 have been fractionated on gels of 3.5–7.5% and 2.5–7.5% acrylamide concentration. In addition to the wide range of fragment sizes which may be run on a single gel, a further advantage of the system is that much sharper bands are obtained compared to conventional constant concentration gels, thus improving resolution.In the molecular-weight range below 5 × 106, for bands whose terminal velocities in the polyacrylamide concentration gradient approach zero, an approximately linear relationship holds between the logarithms of the molecular weights of the fragments and the logarithms of the distances they have migrated in the gel. Thus, by choosing a suitable upper limit to the concentration gradient, the gel system provides a method for estimating approximate molecular weights of unknown DNA fragments, by comparing their mobilities to known standards.
The DNA duplex (designated [A]) corresponding to the nucleotides 1 to 20 of the major yeast alanine transfer RNA (Fig. 1) has been synthesized. The first step involved the T4 ligase-catalyzed joining of d-(5′-32P)-C-C-G-G-A-A-T-C (segment 4, Fig. 1) to the dodecanucleotide, d-(5′-OH)-T-G-G-T-G-G-A-C-G-A-G-T (segment 1, Fig. 1), in the presence of the complementary decanucleotide d-(5′-OH)-C-C-G-G-A-C-T-C-G-T (segment 3, Fig. 1). The resulting icosanucleotide, d-(5′-OH)-T-G-G-T-G-G-A-C-G-A-G-T-C-C-G-G-A-A-T-C, was isolated free from the decanucleotide (segment 3). The synthesis of [A] was then completed by the ligase-catalyzed joining of 5′-32P or 33P-labeled hexanucleotide d-(5′-P)-C-C-A-C-C-A (segment 2) to the 5′-32P or 33P-labeled decanucleotide, d-(5′-P)-C-C-G-G-A-C-T-C-G-T (segment 3), in the presence of the above icosanucleotide.During the above work, the intermediate in the T4 ligase-catalyzed joining was identified as the pyrophosphate formed by the adenylation of the 5′-phosphate group of the oligodeoxynucleotide. Furthermore, the T4 ligase was found to bring about the joining reaction when the hydrogen-bonded duplexes contained mismatched (non-Watson-Crick) base pairs.
Polynucleotide ligase has been purified 1000-fold from extracts of Escherichia coli infected with bacteriophage T4. The enzyme catalyzes (a) the repair of phosphodiester bond scissions introduced into the strands of bihelical DNA by pancreatic DNase and (b) the conversion of hydrogen-bonded circular duplexes or concatemers of λ bacteriophage DNA to covalently bonded circular
molecules or concatemers, respectively. The DNA products of these reactions have been characterized by sedimentation and end
group analysis. The ligase does not catalyze the end to end joining of T7 bacteriophage DNA.
The standard assay for ligase activity measures the protection of a 32P-labeled 5'-phosphoryl group, located at a single strand break in DNA, from attack by bacterial alkaline phosphatase. The
enzyme purification procedure has been redesigned to eliminate the necessity for enzyme assays during the early fractionation
steps and to permit the use of a simple assay, based on ATP-PPi exchange, during the later stages of purification.
The ligase reaction has a specific requirement for a bihelical DNA substrate and for ATP, and it results in the formation
of phosphodiester bonds, AMP, and pyrophosphate. The Km for DNA containing single strand breaks is 1.5 x 10-9 m expressed as the concentration of internal phosphomonoesters. The Km for ATP is 1.4 x 10-5 m, and dATP is a competitive inhibitor of the enzyme (Ki of 3.5 x 10-5 m).
The 23S twisted circular form of ColE(1) DNA has been isolated from Escherichia coli as a tightly associated DNA-protein complex with a sedimentation coefficient of approximately 24S. Treatment of this complex with pronase, trypsin, sodium dodecyl sulfate, Sarkosyl, or heat results in a conversion to a slower sedimenting form of 17S or 18S, as determined by centrifugation in neutral sucrose gradients. These treatments do not alter the sedimentation properties of noncomplexes supercoiled ColE(1) DNA even in the presence of the ColE(1)-protein complex. Electron microscopic analyses indicate that the decrease in sedimentation rate of the ColE(1)-protein complex after treatment with these various agents is largely owing to an induced transition of ColE(1) DNA from the supercoiled to the open circular state.
Bacillus subtilis DNA was heated at constant temperatures within the helix-coil transition interval. Slight increases in the temperature caused the denaturation of greater fractions of DNA. The native molecules were separated from the heat-sensitive ones by means of nitrocellulose chromatography. The fractions thus obtained were characterized for their base composition and ability to hybridize with ribosomal RNA (rRNA). As expected, heat resistance is inversely correlated with thymidine frequency and directly correlated with the content in sequences complementary to rRNA (rDNA).
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