Insertion of nucleotides opposite AP (apurinic/apyrimidinic) sites in DNA during in vitro synthesis: the uniqueness of adenine nucleotides

Technical Report ·

OSTI ID:5715548

Sagher, D; Strauss, B

M13 DNA containing 20 to 30 AP (apurinic/apyrimidinic) sites per intact circular modecule was prepared by growing phage on an ung/sup -/ dut/sup -/ E. coli mutant and treating the DNA with uracil-N-glycosylase. AP sites obstruct in vitro DNA synthesis catalyzed by E. coli pol I. The position at which termination of synthesis occurs was determined for four enzymes. T4 DNA polymerase terminates one nucleotide before putative AP sites. DNA pol I, AMV reverse transcriptase and DNA polymerase alpha terminate synthesis either before or at the site of an AP lesion depending on the particular sequence. We determine the identity of the nucleotide inserted opposite AP site by synthesizing up to the lesion in a first stage reaction using T4 DNA polymerase and then determining elongation in a second stage. Purines are inserted opposite AP sites more readily than pyrimidines and dATP is more efficient than dGTP in promoting such elongation. The DNA-dependent conversion of dNTP to dNMP was determined in mixtures of all four dNTP's using AP DNA. The production of dAMP from dATP occurs most readily. We conclude that there is an inherent specificity for the incorporation of A opposite AP sites in this in vitro system. Insofar as the model system reflects in vivo mutational events, our data suggest that depurination should produce transversions and depyrimidination, transitions.

• Research Organization: Chicago Univ., IL (USA). Dept. of Microbiology
• DOE Contract Number: AC02-76EV02040
• OSTI ID: 5715548
• Report Number(s): DOE/EV/02040-15; ON: DE84001269
• Country of Publication: United States
• Language: English

Related Subjects

550200* -- Biochemistry
59 BASIC BIOLOGICAL SCIENCES
BACTERIA
BACTERIOPHAGES
BIOSYNTHESIS
DNA
DNA REPLICATION
DNA-ASE
ENZYMES
ESCHERICHIA COLI
ESTERASES
HYDROLASES
IN VITRO
MICROORGANISMS
MUTANTS
NUCLEIC ACID REPLICATION
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PARASITES
PHOSPHODIESTERASES
SYNTHESIS
TRANSCRIPTION
VIRUSES