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Volumn 10, Issue 9, 2000, Pages 907-910

Incorporation of 4-thiothymidine into DNA by the Klenow fragment and HIV-1 reverse transcriptase

Author keywords

[No Author keywords available]

Indexed keywords

RNA DIRECTED DNA POLYMERASE;

EID: 0034193403     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0960-894X(00)00123-2     Document Type: Article
Times cited : (14)

References (34)
  • 10
    • 84992267203 scopus 로고    scopus 로고
    • note
    • -1 (ref 4d).
  • 21
    • 84992255528 scopus 로고    scopus 로고
    • 260 nm using the program Oligo (National Biosciences, Plymouth, MN)
    • 260 nm using the program Oligo (National Biosciences, Plymouth, MN).
  • 22
    • 84992245217 scopus 로고    scopus 로고
    • The value of Δε was obtained by incubating 25 μM template-primer (DNA-9-mer/DNA-20-mer) and excess 4S-TTP with exonuclease-free Klenow fragment (Unites States Biochemical) under standard buffer conditions (ref. 13) and allowing the reaction to go to completion. Exonuclease-free Klenow fragment was used since with intact Klenow fragment the total absorbance change was smaller and changes in absorbance were observed to reverse direction with prolonged reaction times, indicating slow hydrolysis of the product
    • The value of Δε was obtained by incubating 25 μM template-primer (DNA-9-mer/DNA-20-mer) and excess 4S-TTP with exonuclease-free Klenow fragment (Unites States Biochemical) under standard buffer conditions (ref. 13) and allowing the reaction to go to completion. Exonuclease-free Klenow fragment was used since with intact Klenow fragment the total absorbance change was smaller and changes in absorbance were observed to reverse direction with prolonged reaction times, indicating slow hydrolysis of the product.
  • 23
    • 84992245231 scopus 로고    scopus 로고
    • Note The following three template DNA strands were used: 5′-AAACCCTTGAACGGCTGCGA-OH, 5′-AAACCCTTAAACGGCTGCGA-OH, and 5′-AAACCCTAAAACGGCTGCGA-OH, which when annealed with primer 9-mer in the final template-primer duplex contain sites for incorporation of 2, 3 and 4 (thio)thymidines, respectively, opposite to the underlined A's
    • The following three template DNA strands were used: 5′-AAACCCTTG AA CGGCTGCGA-OH, 5′-AAACCCTT AAA CGGCTGCGA-OH, and 5′-AAACCCT AAAA CGGCTGCGA-OH, which when annealed with primer 9-mer in the final template-primer duplex contain sites for incorporation of 2, 3 and 4 (thio)thymidines, respectively, opposite to the underlined A's.
  • 25
    • 84992249669 scopus 로고    scopus 로고
    • 2, 1 mM DTT and 0.5 mM EGTA, as used for previous kinetic experiments with Klenow fragment and similar template-primer duplexes (ref 1c). Kinetic parameters (25 °C) were obtained by fitting the Michaelis-Menten equation to initial rates measured at several triphosphate concentrations using the program Enzfitter (Biosoft, Cambridge, UK)
    • 2, 1 mM DTT and 0.5 mM EGTA, as used for previous kinetic experiments with Klenow fragment and similar template-primer duplexes (ref 1c). Kinetic parameters (25 °C) were obtained by fitting the Michaelis-Menten equation to initial rates measured at several triphosphate concentrations using the program Enzfitter (Biosoft, Cambridge, UK).
  • 26
    • 84992238783 scopus 로고    scopus 로고
    • (a) Stopped-flow spectrometers from Kin-Tek Instruments (ref 14b), with two identical loading syringes (actuated by either nitrogen gas pressure or a stepping motor) leading to a rapid (˜1.6 ms) mixing chamber connected to a stop syringe, were used. The stop chamber was irradiated by a variable wavelength monochromatic light source set to 340 nm and monitored by a photodiode connected to a computer. Syringe A contained 4 μM Klenow fragment and 20 μM DNA-9-mer/DNA-20-mer (ref 9) in MOPS buffer. Syringe B contained 5-60 μM 4S-TTP in MOPS buffer. Data were collected for time periods ranging from 0.1 s to up to 10 s. The results of several runs at each substrate concentration were averaged
    • (a) Stopped-flow spectrometers from Kin-Tek Instruments (ref 14b), with two identical loading syringes (actuated by either nitrogen gas pressure or a stepping motor) leading to a rapid (˜1.6 ms) mixing chamber connected to a stop syringe, were used. The stop chamber was irradiated by a variable wavelength monochromatic light source set to 340 nm and monitored by a photodiode connected to a computer. Syringe A contained 4 μM Klenow fragment and 20 μM DNA-9-mer/DNA-20-mer (ref 9) in MOPS buffer. Syringe B contained 5-60 μM 4S-TTP in MOPS buffer. Data were collected for time periods ranging from 0.1 s to up to 10 s. The results of several runs at each substrate concentration were averaged.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.