Open AccessDNA synthesis in vivo and in vitro in Escherichia coli irradiated with ultraviolet light
Author(s) -
BANFALVI Gaspar,
SLEZARIKOVA Viera,
SEDLIAKOVA Milena,
ANTONI Ferenc
Publication year - 1987
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1987.tb10601.x
Subject(s) - dna synthesis , biology , novobiocin , dna , escherichia coli , microbiology and biotechnology , biochemistry , nucleic acid , ribonucleoside , ultraviolet light , rna , chemistry , photochemistry , gene , antibiotics
DNA synthesis was followed in vivo and in permeable Escherichia coli after (a) ultraviolet light irradiation, (b) irradiation and incubation in a growth medium containing chloramphenicol and (c) in unirradiated cells. In vitro , replicative type DNA synthesis was partially restored after incubation of cells in medium containing chloramphenicol, but not in vivo . The DNA was pulse‐labeled in permeable cells in the presence of deoxyribonucleoside triphosphates and ribonucleoside triphosphates. dCTP was replaced by 5‐Hg‐dCTP as a substrate for DNA synthesis. Hg‐DNA was separated from cellular nucleic acids on thiol‐agarose affinity columns. The 5′ termini of newly synthesized DNA were analyzed after treatment with alkaline phosphatase and rephosphorylation with polynucleotide kinase and [γ‐ 32 P]ATP. DNA synthesis in unirradiated permeable E. coli represents a replicative process dependent on ATP and inhibited by novobiocin. About 70% of the nascent DNA carried terminally labeled RNA moiety at its 5'end. In vitro DNA synthesis in irradiated cells was suppressed and hardly influenced by the presence of ATP or novobiocin. The 5′‐RNA content of this cell population was less than 5%.