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[8] Repair of overlapping DNA termini
[8]
REPAIR OF OVERLAPPING DNA
TERMINI
63
[8] R e p a i r o f O v e r l a p p i n g D N A T e r m i n i
By HOWARD M. GOODMAN The natural cohesive termini of bacteriophage DNA 1 such as h and the staggered phosphodiester bond cleavages produced by various restriction endonucleases 2 could have several possible arrangements) If the arrangement is such that a protruding 5' single-stranded end and an internal 3'-hydroxyl end are formed, then the termini will serve both as primer and template for repair synthesis by a DNA polymerase, l'a Method
Principle. Overlapping DNA termini can be repaired using the RNAdirected DNA polymerase ("reverse transcriptase") from RNA tumor viruses and radioactive nucleoside triphosphates. The incorporation into the termini can be quantified and the sequence analyzed from nearest neighbor data. Labeling Procedure. The DNA to be analyzed is digested with the appropriate restriction endonuclease 2 (if required), extracted with an equal volume of phenol-chloroform [DNA:phenoI:CHCla(1 : 1 : 1)] and the DNA precipitated with 2 volumes of 95% ethanol after making the aqueous phase 0.3 M in sodium acetate. After chilling ( - 7 0 ° for 30 min), the precipitate is collected by centrifugation and washed once with 95% ethanol. The DNA is then dissolved in a small volume of water (see below). These operations are easily performed in 1.5 ml Eppendorf Micro test tubes which can be centrifuged in an Eppendorf Microcentrifuge. The overlapping termini are radioactively labeled in a reaction which contains the following ingredients in a total volume of 25/~1: 0,1 to 2 pmole of termini, 0.1 M Tris-HC1 (pH 8.0), 2 mM 2-mercaptoethanol, 10 mM MgCh, 5-10 /zM dNTP (various combinations of nonradioactive and [a-zzP]-labeled nucleoside triphosphates, specific activities 100-1000 Ci/ raM), and 0.2 munits of reverse transcriptase per microgram of DNA (specific activity about 0.1 unit//zg). 4 The reaction mixture is incubated at 37° for 2 hr. The reaction is stopped by addition of 0.4 ml of 0.05M sodium pyrophosphate (NaPPi) plus 0.05 M Na2 EDTA and 0.2 ml calf thymus R. W u and E. Taylor, J. Mol. Biol. 57, 491 (1971). 2 R. J. Roberts, Crit. Rev. Biochem. 3, 123 (1976). a j. H e d g p e t h , H. M. G o o d m a n , and H. W. Boyer, Proc. Natl. A c a d . Sci. U . S . A . 69, 3448 (1972). A. J. Faras, J. M. Taylor, J. P. McDonnell, W. E. L e v i n s o n , a n d J. M. Bishop, Biochemistry 11, 2334 (1972).
METHODS IN ENZYMOLOGY,VOL. 65
Copyright © 1980by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181965-5
64
TECHNIQUES FOR LABELING TERMINI
[8]
DNA (400/~g/ml) as carrier. Ten percent (60/~1) of the reaction is diluted into 0.2 ml NaPPI plus 0.2 ml calf thymus DNA (400/~g/ml) for the determination of total incorporation. After precipitation with 1 ml of 7% perchloric acid containing 0.1 M NaPPi and standing on ice for 30 min, the precipitate is collected on glass fiber filters and counted. Nearest Neighbor Analysis. The remainder of the reaction (90%; 565/xl) is extracted with an equal volume (600/~l) of phenol and the aqueous phase containing the labeled DNA dialyzed for 2 days against at least three changes of 4 liters of 0.3 M sodium acetate and 10 mM Tris-HCl (pH 7.5). (Any other method, such as gel filtration, which removes unincorporated nucleoside triphosphates can be substituted for the dialysis step.) The DNA is then precipitated with 2 volumes of 95% ethanol and resuspended in a minimal volume of water. The DNA is then digested to completion by first digesting with 40 units of micrococcal nuclease in 10 mM sodium borate buffer (pH 8.6) plus 20 mM CaCl~ for 30 rain at 37° and then with 0.2 units of spleen phosphodiesterase in 50 mM Tris-HCl (pH 7.5), 10 mM MgC12, and 5 mM rAMP. The 3'-mononucleotides produced are separated by high-voltage electrophoresis (3-5 kV, 1-3 hr) on Whatman 3 MM paper in pyridine-acetate buffer, pH 3.5. Appropriate absorbance markers can be added where necessary, the pyridine removed in NH3 vapor, and after drying the paper the makers are visible under a short-wave uv lamp. The incorporated radioactivity can be quantitated by cutting-out and counting the paper in an end-window Geiger counter or scintillation counter and/or visualized by autoradiography using X-ray film. Comme~s It is useful to optimize the incorporation reactions prior to performing quantitative studies by varying the time of incubation, the enzymetemplate ratio, and the concentrations of nucleoside triphosphates. The method has been successfully used in determining the cleavage site for the E c o R I and E c o R I I restriction endonucleases? ,5 The reader may also want to consult a previous article in this series by Wu et al. s Acknowledgment This work was supported in part by a grant from the United States Public Health Service
(CA 14026). 5 H. W. Boycr, L. T. Chow, A. Dugaiczyk, J. Hedgpeth, and H. M. Goodman, Nature (London) 244, 40 (1973). s R. Wu, R. Padmanabhan, and R. Bambara, this series, Vol. 29, p. 231.
REPAIR OF OVERLAPPING DNA
TERMINI
63
[8] R e p a i r o f O v e r l a p p i n g D N A T e r m i n i
By HOWARD M. GOODMAN The natural cohesive termini of bacteriophage DNA 1 such as h and the staggered phosphodiester bond cleavages produced by various restriction endonucleases 2 could have several possible arrangements) If the arrangement is such that a protruding 5' single-stranded end and an internal 3'-hydroxyl end are formed, then the termini will serve both as primer and template for repair synthesis by a DNA polymerase, l'a Method
Principle. Overlapping DNA termini can be repaired using the RNAdirected DNA polymerase ("reverse transcriptase") from RNA tumor viruses and radioactive nucleoside triphosphates. The incorporation into the termini can be quantified and the sequence analyzed from nearest neighbor data. Labeling Procedure. The DNA to be analyzed is digested with the appropriate restriction endonuclease 2 (if required), extracted with an equal volume of phenol-chloroform [DNA:phenoI:CHCla(1 : 1 : 1)] and the DNA precipitated with 2 volumes of 95% ethanol after making the aqueous phase 0.3 M in sodium acetate. After chilling ( - 7 0 ° for 30 min), the precipitate is collected by centrifugation and washed once with 95% ethanol. The DNA is then dissolved in a small volume of water (see below). These operations are easily performed in 1.5 ml Eppendorf Micro test tubes which can be centrifuged in an Eppendorf Microcentrifuge. The overlapping termini are radioactively labeled in a reaction which contains the following ingredients in a total volume of 25/~1: 0,1 to 2 pmole of termini, 0.1 M Tris-HC1 (pH 8.0), 2 mM 2-mercaptoethanol, 10 mM MgCh, 5-10 /zM dNTP (various combinations of nonradioactive and [a-zzP]-labeled nucleoside triphosphates, specific activities 100-1000 Ci/ raM), and 0.2 munits of reverse transcriptase per microgram of DNA (specific activity about 0.1 unit//zg). 4 The reaction mixture is incubated at 37° for 2 hr. The reaction is stopped by addition of 0.4 ml of 0.05M sodium pyrophosphate (NaPPi) plus 0.05 M Na2 EDTA and 0.2 ml calf thymus R. W u and E. Taylor, J. Mol. Biol. 57, 491 (1971). 2 R. J. Roberts, Crit. Rev. Biochem. 3, 123 (1976). a j. H e d g p e t h , H. M. G o o d m a n , and H. W. Boyer, Proc. Natl. A c a d . Sci. U . S . A . 69, 3448 (1972). A. J. Faras, J. M. Taylor, J. P. McDonnell, W. E. L e v i n s o n , a n d J. M. Bishop, Biochemistry 11, 2334 (1972).
METHODS IN ENZYMOLOGY,VOL. 65
Copyright © 1980by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181965-5
64
TECHNIQUES FOR LABELING TERMINI
[8]
DNA (400/~g/ml) as carrier. Ten percent (60/~1) of the reaction is diluted into 0.2 ml NaPPI plus 0.2 ml calf thymus DNA (400/~g/ml) for the determination of total incorporation. After precipitation with 1 ml of 7% perchloric acid containing 0.1 M NaPPi and standing on ice for 30 min, the precipitate is collected on glass fiber filters and counted. Nearest Neighbor Analysis. The remainder of the reaction (90%; 565/xl) is extracted with an equal volume (600/~l) of phenol and the aqueous phase containing the labeled DNA dialyzed for 2 days against at least three changes of 4 liters of 0.3 M sodium acetate and 10 mM Tris-HCl (pH 7.5). (Any other method, such as gel filtration, which removes unincorporated nucleoside triphosphates can be substituted for the dialysis step.) The DNA is then precipitated with 2 volumes of 95% ethanol and resuspended in a minimal volume of water. The DNA is then digested to completion by first digesting with 40 units of micrococcal nuclease in 10 mM sodium borate buffer (pH 8.6) plus 20 mM CaCl~ for 30 rain at 37° and then with 0.2 units of spleen phosphodiesterase in 50 mM Tris-HCl (pH 7.5), 10 mM MgC12, and 5 mM rAMP. The 3'-mononucleotides produced are separated by high-voltage electrophoresis (3-5 kV, 1-3 hr) on Whatman 3 MM paper in pyridine-acetate buffer, pH 3.5. Appropriate absorbance markers can be added where necessary, the pyridine removed in NH3 vapor, and after drying the paper the makers are visible under a short-wave uv lamp. The incorporated radioactivity can be quantitated by cutting-out and counting the paper in an end-window Geiger counter or scintillation counter and/or visualized by autoradiography using X-ray film. Comme~s It is useful to optimize the incorporation reactions prior to performing quantitative studies by varying the time of incubation, the enzymetemplate ratio, and the concentrations of nucleoside triphosphates. The method has been successfully used in determining the cleavage site for the E c o R I and E c o R I I restriction endonucleases? ,5 The reader may also want to consult a previous article in this series by Wu et al. s Acknowledgment This work was supported in part by a grant from the United States Public Health Service
(CA 14026). 5 H. W. Boycr, L. T. Chow, A. Dugaiczyk, J. Hedgpeth, and H. M. Goodman, Nature (London) 244, 40 (1973). s R. Wu, R. Padmanabhan, and R. Bambara, this series, Vol. 29, p. 231.