Analysis of the Restoration of DNA Integrity in S-Phase and non-S-Phase Cells after the Treatment with Methyl Methanesulfonate by DNA Comet Assay

The extent and nature of DNA damage, as well as mechanisms for restoration of genome integrity under conditions of genotoxic stress caused by alkylation of DNA bases can differ significantly in S-phase and non-S-phase cells. In this work, we used the alkaline DNA comet assay after pulse labeling of replicating DNA with 5-iodo-2'-deoxyuridine (IdU) to analyze DNA integrity restoration in S-phase and non-S-phase human dermal fibroblasts exposed to DNA-alkylating agent methyl methanesulfonate (MMS). Quantitative analysis of IdU-labeled DNA comets enables to assess both the efficiency of base excision repair (BER) in replicating genome regions and the extent of restoration of normal DNA replication in individual S-phase cells after MMS treatment. At the same time, additional staining of DNA comets with SYBR Gold can be used to compare the efficiency of the whole-genome BER in IdU-labeled S-phase cells and in non-S-phase cells that do not contain IdU label.