Efficient synthesis of supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication
The authors report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, they report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. They demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.
- Research Organization:
- Univ. of Texas, Austin (USA)
- OSTI ID:
- 7130773
- Journal Information:
- Biochemistry; (United States), Vol. 27:9
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
DNA
BIOLOGICAL RADIATION EFFECTS
BIOSYNTHESIS
CONFORMATIONAL CHANGES
DNA ADDUCTS
RADIOINDUCTION
ATP
DNA REPLICATION
ELECTROPHORESIS
IN VITRO
NEAR ULTRAVIOLET RADIATION
PHOSPHORUS 32
PSORALEN
ADDUCTS
ANTICOAGULANTS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL EFFECTS
COUMARINS
DAYS LIVING RADIOISOTOPES
DRUGS
ELECTROMAGNETIC RADIATION
HEMATOLOGIC AGENTS
HETEROCYCLIC COMPOUNDS
ISOTOPES
LIGHT NUCLEI
NUCLEI
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
PHOSPHORUS ISOTOPES
RADIATION EFFECTS
RADIATIONS
RADIOISOTOPES
SYNTHESIS
ULTRAVIOLET RADIATION
560120* - Radiation Effects on Biochemicals
Cells
& Tissue Culture