2013 Volume 35 Issue 2 Pages 46-52
The comet assay can detect single-strand breaks (SSBs) as initial lesions and those that developed from alkali-labile sites under alkaline conditions (pH>12.6). While the incision of UV-induced pyrimidine dimers creates a long gap of about 30 bp during nucleotide excision repair (NER) that is re-synthesized and repaired by DNA polymerase δ/ε, base excision repair (BER) creates a short gap of 1-6 bp that is closed by DNA polymerase β. Some chemicals are known to affect the formation of comet tails by the inhibition of enzymes taking part in DNA repair pathways. In this study, we investigated how 2′,3′-dideoxythymidine (ddT) which is a precursor of DNA polymerase β inhibitor, 2′,3′-dideoxythymidine 5′-triphosphate (ddTTP), affects comet-tail formation. The effects of three kinds of DNA re-synthesis inhibitors on the response of the comet assay were studied in TK+/− heterozygotes of TK6 human lymphoblastoid cells. Aphidicolin (APC) and the combination of cytosine-1-β-D-arabinofuranoside and hydroxyurea (araC/HU) enhanced comet-tail formation by UV, a UV-mimetic agent, 4-nitroquinoline-1-oxide (4NQO), and methylating agents, methyl methanesulfonate (MMS) and methyl nitrosourea (MNU). On the other hand, ddT enhanced comet-tail formation by MMS and MNU, but not UV and 4NQO. Since ddT did not affect comet-tail formation by MMS and MNU in TK−/− TK6 cells, ddTTP which is expected to be formed from ddT by thymidine kinase in TK+/− TK6 cells would inhibit the resealing of short gaps left by excision of damaged bases induced by MMS and MNU. Taking into consideration that 75% of BER was estimated to be due to DNA polymerase β in human cells, ddT can enhance a comet-positive response upon exposure to mutagens that produce damaged bases removed by BER, showing that BER depending on DNA polymerase β can be distinguished from NER by the enhancement of a comet-positive response by ddT.
