REV1 Coordinates a Multi-Faceted Tolerance Response to DNA Alkylation Damage and Prevents Chromosome Shattering in Drosophila melanogaster

Abstract

When replication forks encounter damaged DNA, cells utilize DNA damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses following alkylation damage in Drosophila melanogaster. We report that translesion synthesis, rather than template switching, is the preferred response to alkylation-induced damage in diploid larval tissues. Furthermore, we show that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Drosophila larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of {gamma}-H2Av foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerases eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, our results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability. Author SummaryOrganisms have evolved several ways to continue copying their DNA when it is damaged, grouped into the categories of translesion synthesis and template switching. These damage tolerance mechanisms prevent replication forks from collapsing when they encounter DNA damage and prevent catastrophic genome instability and cell death. While the proteins and pathways involved in damage tolerance are beginning to be understood at the single cell level, how they are regulated in multicellular organisms is an intriguing question. In this study, we investigated the mechanisms by which Drosophila tolerate alkylation damage during their development. We discovered that tissues containing rapidly dividing diploid cells favor translesion synthesis over template switching, preferentially utilizing different translesion polymerases in a context-dependent manner. Furthermore, we showed that the REV1 protein, best known for its role in recruiting translesion DNA polymerases to damage sites, performs multiple functions during damage tolerance. Together, our results demonstrate that damage tolerance preferences for multicellular organisms may differ from those observed in cultured cells, and establish Drosophila as a useful model system for studying tolerance mechanisms.