Pyrimidine Dimer Formation and Oxidative Damage in M13 Bacteriophage Inactivation by Ultraviolet C Irradiation¶

Abstract

The mechanism by which UV‐C irradiation inactivates M13 bacteriophage was studied by analyzing the M13 genome using agarose gel electrophoresis and South‐Western blotting for pyrimidine dimers. The involvement of singlet oxygen (1O2) was also investigated using azide and deuterium oxide and under deoxygenated conditions. With a decrease in M13 infectivity on irradiation, single‐stranded circular genomic DNA (sc‐DNA) was converted to Form I and Form II, which had an electrophoretic mobility between that of sc‐DNA and linear‐form DNA. However, the amount of sc‐DNA remaining was not correlated with the survival of M13. The formation of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts ((6–4)PP) increased as a function of irradiation dose. The decrease in M13 infectivity was highly correlated with the increase in CPD and (6–4)PP, whereas no change was seen in M13 coat protein on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. 8‐Oxo‐7,8‐dihydro‐2′‐deoxyguanosine did not form in the M13 genome after UV‐C irradiation. Inactivation of M13 was neither enhanced by deuterium oxide nor inhibited by azide. Deoxygenation of the M13 suspension did not affect the inactivation, indicating that 1O2 did not participate in the inactivation of M13 by UV‐C irradiation under these conditions. These results indicated that UV‐C irradiation induced not only CPD and (6–4)PP formation but also additional tertiary structural change in DNA inside the M13 virions, resulting in primary damage and a loss of infectivity. The indirect effect of UV‐C irradiation such as 1O2 production followed by oxidative damage to nucleic acids and proteins might have contributed less, if at all, to the inactivation of M13 than the direct effect of UV‐C.

Microselection - affinity selecting antibodies against a single Rare cell in a heterogeneous population

“…For the phage particle to be able to replication, the phage DNA must be capable to replicate in the host bacteria, as previously been shown by phage inactivation by UV irradiation [14] Fig. S1, and in the cartoon in Fig.S2.…”

Subtle Differences in Virus Composition Affect Disinfection Kinetics and Mechanisms

“…This is supported by the work of Napolitano et al, who suggested that inactivation should be due to protein damage, as ClO 2 reacts more readily with amino acids than with nucleotides (11). UV is often reported to cause fatal genome damage by forming pyrimidine dimers (4,12,13), and yet protein damage has also been reported (14,15). Singlet oxygen has been found to cause protein crosslinking (16), whereas other studies report genome damage as the main target for disinfection (17,18).…”

Inactivation of Poliovirus 1 and F-Specific RNA Phages and Degradation of Their Genomes by UV Irradiation at 254 Nanometers

“…We monitored genome degradation using reverse transcription (RT)-PCR, because with this technique the viral genome must undergo enzymatic transcription before detection. For the considered viruses, detection of viral RNA would thus decline with increasing fluence whatever the targeted fragment, a rationale previously used for DNA of irradiated phage M13 (16). Hence, the decrease of viral infectivity due to UV was monitored simultaneously with the effect of UV radiation on nucleic acids.…”