Chloramphenicol effects on DNA replication in UV-damaged bacteria

Abstract

Increasing UV-doses to cultures of Escherichia coli strain B/r decreased progressively the amount of DNA which was formed in the presence of chloramphenicol (160 μg/ml) from the amount formed in unirradiated control cultures in chloramphenicol-containing medium. This is attributed to the progressive inactivation of active sites of DNA replication by UV. In order to form DNA the bacteria must then replicate from the chromosomal fixed origin, an activity which requires protein synthesis and thus cannot occur in the presence of chloramphenicol. Such damage was shown to be subject to photoreactivation after lower UV-doses and thus is the pyrimidine dimer. After higher doses non-photoreversible lesions began to accumulate so that all such damage became non-photoreversible after 96 erg/mm 2. The rate of synthesis of DNA in the presence of chloramphenicol was shown to be very close to the rate shown by bacteria incubated in the absence of chloramphenicol, indicating that all active sites of replication remaining after UV-damage remain active in the presence of chloramphenicol, as expected if the limiting effect of chloramphenicol is on initiation at the chromosomal origin and not due to reduction in rate of DNA replication. A much lower concentration of chloramphenicol (2 μg/ml) blocking only the chloramphenicol-sensitive event in control of DNA replication described by Ward and Glaser 15, imposed a limitation in DNA accumulation in the culture of somewhat less than a doubling, as would be expected if the antibiotic at this concentration does not block the chloramphenicol-resistant control event. DNA degradation occured with incubation of bacteria given a UV-dose sufficient to inactivate all active DNA replication sites on their chromosomes, when in medium containing chloramphenicol concentrations (above 20 μg/ml) sufficient to block the chloramphenicol-resistant control event. Such breakdown resulted in death. The damage responsible for such death and DNA breakdown was not photoreversible after this dose, supporting the hypothesis that breakdown results from non-photoreversible inactivation of active DNA replication sites. This was in contrast to increased death in UV-damaged bacteria promoted by nalidixic acid, a specific inhibitor of DNA replication, which could be prevented in part by light exposure after the same UV-dose.