Enzymatic Breakage and Joining of Deoxyribonucleic Acid

Harvey, Clifford; Gabriel, Thomas F.; Wilt, Elaine M.; Richardson, Charles C.

doi:10.1016/s0021-9258(18)62043-5

Cited by 70 publications

(5 citation statements)

References 28 publications

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“…4). Our observation that adenylation of DNA ligase occurs in the presence of dATP or thio dATP, coupled with the fact that the chemically generated DNA-deoxyadenylate (DNA-dAMP) sustains the phosphodiester-bond formation with release of dAMP [13], suggests that dATP exerts its major inhibitory effect upon the second step of DNA ligation, that is, the formation ofthe complex involving DNA-Mg2", AMP and ligase, perhaps by stabilizing the dAMP-enzyme intermediate and preventing the adenylyl transfer from ligase to DNA.…”

Section: Resultsmentioning

confidence: 93%

Use of ATP, dATP and their α-thio derivatives to study DNA ligase adenylation

Montecucco

Lestingi

Pedrali-Noy

et al. 1990

Biochemical Journal

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Bacteriophage-T4 and human type I DNA ligases were found capable of self-adenylating upon exposure to both ribo- and deoxyribo-[alpha-35S]thio-ATP. However, the joining reaction does not take place in the presence of the deoxyribotriphosphates. Enzyme adenylation is reversed in all cases by an excess of PPi, but the rate of reversion is lower with thio derivatives. Therefore thio derivatives can be used to study the adenylation of DNA ligases and to search for specific inhibitors of the first step of the ligation reaction. In addition we show that thio derivatives can be used to detect DNA ligase adenylation activity covalently bound to a solid matrix.

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Section: Resultsmentioning

confidence: 93%

Use of ATP, dATP and their α-thio derivatives to study DNA ligase adenylation

Montecucco

Lestingi

Pedrali-Noy

et al. 1990

Biochemical Journal

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“…nating 3,-d(AT)BxC4(AT)"-3' (n = 4-7) oligonucleotides with good efficiencies (lanes D-G), and the products showed different mobilities than for the ligation product (30-mer) of R-l to unphosphorylated R-l (lane B). The fastest moving band that is visible in all ligation reactions corresponds to the AMP-activated ligation intermediate of R-l which accumulates at low reaction temperatures (Harvey et al, 1971). The ligation products of the R-l hairpin and 3/-d(AT)^cC4(AT)"-3/ have the gel electrophoretic mobility of (19 + 4n)-mers that are the result of the addition of one R-l hairpin to one 3'-3' alternating AT oligonucleotide.…”

Section: Resultsmentioning

confidence: 99%

Length dependent formation of parallel-stranded DNA in alternating AT segments

Germann¹,

Bw²,

Rt³

et al. 1990

Biochemistry

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Parallel-stranded DNA can be formed from alternating AT segments and is not restricted exclusively to homooligomeric AT sequences. DNA oligonucleotides 3'-d(AT)nxC4(AT)n-3' (where x indicates the location of the 5'-5' phosphodiester linkage) form parallel-stranded hairpin structures at micromolar strand concentration for n = 4 or 5 but not for n = 6, 7. The spectral properties of the parallel-stranded structures are similar to those of the hairpin structures containing homooligomeric AT stems. However, parallel-stranded structures formed in alternating AT segments are significantly less stable than either their corresponding antiparallel control or the homooligomeric parallel AT hairpins as evidenced by their lower helix-coil transition enthalpy, melting temperature, and stability constant. This results in a remarkable polymorphism which is most pronounced for 3'-d(AT)5xC4(AT)5-3'. This oligonucleotide can exist as a parallel-stranded hairpin, coil, or concatameric antiparallel structure(s), depending on temperature and strand concentration. These results suggest simple guidelines for the design of parallel-stranded DNA. In addition, we present a model for the assessment of the stability of parallel-stranded duplex structures formed from AT base pairs based on their sequence.

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“…The effects of ATP concentration upon the rate and yield of the reaction can be rationalized in terms of the mechanism. Since by analogy to DNA ligase, free enzyme is probably required for the third step in the reaction (Harvey et al, 1971), high ATP levels lower its rate by sequestering the enzyme in the adenylylated form. However, in the presence of an excess of pdNp over ATP, free enzyme is regenerated by the rapid formation of the adenylylated deoxyribonucleoside bisphosphate intermediate in step 2 (Figure 4), High levels of ATP, therefore, decrease the rate of reaction by lowering the concentration of free enzyme but increase the yield by maintaining a high concentration of the activated donor required in step 3.…”

Section: Discussionmentioning

confidence: 99%

T4 RNA ligase joins 2'-deoxyribonucleoside 3',5'-bisphosphates to oligodeoxyribonucleotides

Hinton¹,

Báez²,

Gumport³

1978

Biochemistry

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T4 RNA ligase catalyzes the ATP-dependent addition of a single 2'-deoxyribonucleoside 3',5'-bisphosphate to the 3'-hydroxyl of an oligodeoxyribonucleotide. The bisphosphate is joined to the deoxyoligomer by a 3' leads to 5' phosphodiester bond and the product, which is terminated by a 3'-phosphate, is one nucleotide longer than the substrate. Bisphosphates of dAdo, dCyd, dGuo, dThd, and dUrd are donors and oligodeoxyribonucleotides with dA, dC, dG, dT, or dU 3' termini act as acceptors. The preferred residue for both donor and acceptor is dCyd. Deoxyoligomers from 3 to 12 residues in length are active as acceptors. To obtain good yields, high concentration of enzyme, long incubation time at low temperature, and manganous rather than Mg(II) ion are required. Under optimal conditions, yields calculated with respect to deoxyoligomer converted to product vary from 40 to greater than 95%. The turnover number of the enzyme for DNA joining is extremely low but, because the preparation is nearly free of DNases, there is less than 3% degradation of substrate or product after 6 days of reaction. We anticipate that this reaction will serve as the basis for a method for the stepwise enzymatic synthesis of DNA of defined sequence.

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Enzymatic Breakage and Joining of Deoxyribonucleic Acid

Cited by 70 publications

References 28 publications

Use of ATP, dATP and their α-thio derivatives to study DNA ligase adenylation

Use of ATP, dATP and their α-thio derivatives to study DNA ligase adenylation

Length dependent formation of parallel-stranded DNA in alternating AT segments

T4 RNA ligase joins 2'-deoxyribonucleoside 3',5'-bisphosphates to oligodeoxyribonucleotides

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