Replication Stress Induces Micronuclei Comprising of Aggregated DNA Double-Strand Breaks

Background: Micronuclei (MN) in mammalian cells serve as a reliable biomarker of genomic instability and genotoxic exposure. Elevation of MN is commonly observed in cells bearing intrinsic genomic instability and in normal cells exposed to genotoxic agents. DNA double-strand breaks are marked by phosphorylation of H2AX at serine 139 (gamma-H2AX). One subclass of MN contains massive and uniform gamma-H2AX signals. This study tested whether this subclass of MN can be induced by replication stress. Principal Findings: We observed that a large proportion of MN, from 20% to nearly 50%, showed uniform staining by antibodies against gamma-H2AX, a marker of DNA double-strand breaks (DSBs). Such micronuclei were designated as MN-gamma-H2AX (+). We showed that such MN can be induced by chemicals that are known to cause DNA replication stress and S phase arrest. Hydroxyurea, aphidicolin and thymidine could all significantly induce MN-gamma-H2AX (+), which were formed during S phase and appeared to be derived from aggregation of DSBs. MN-gamma-H2AX (-), MN that were devoid of uniform gamma-H2AX signals, were induced to a lesser extent in terms of fold change. Paclitaxel, which inhibits the disassembly of microtubules, only induced MN-gamma-H2AX (-). The frequency of MN-gamma-H2AX (+), but not that of MN-gamma-H2AX (-), was also significantly increased in cells that experience S phase prolongation due to depletion of cell cycle regulator CUL4B. Depletion of replication protein A1 (RPA1) by RNA interference resulted in an elevation of both MN-gamma-H2AX (+) and MN-gamma-H2AX (-). Conclusions/Significance: A subclass of MN, MN-gamma-H2AX (+), can be preferentially induced by replication stress. Classification of MN according to their gamma-H2AX status may provide a more refined evaluation of intrinsic genomic instabilities and the various environmental genotoxicants.