Exploring RAD18-dependent replication of damaged DNA and discontinuities: A collection of advanced tools
Journal of biotechnology(2024)
摘要
DNA damage tolerance (DDT) pathways mitigate the effects of DNA damage during replication by rescuing the replication fork stalled at a DNA lesion or other barriers and also repair discontinuities left in the newly replicated DNA. From yeast to mammalian cells, RAD18-regulated translesion synthesis (TLS) and template switching (TS) represent the dominant pathways of DDT. Monoubiquitylation of the polymerase sliding clamp PCNA by HRAD6A-B/RAD18, an E2/E3 protein pair, enables the recruitment of specialized TLS polymerases that can insert nucleotides opposite damaged template bases. Alternatively, the subsequent polyubiquitylation of monoubiquitin-PCNA by Ubc13-Mms2 (E2) and HLTF or SHPRH (E3) can lead to the switching of the synthesis from the damaged template to the undamaged newly synthesized sister strand to facilitate synthesis past the lesion. When immediate TLS or TS cannot occur, gaps may remain in the newly synthesized strand, partly due to the repriming activity of the PRIMPOL primase, which can be filled during the later phases of the cell cycle. The first part of this review will summarize the current knowledge about RAD18-dependent DDT pathways, while the second part will offer a molecular toolkit for the identification and characterization of the cellular functions of a DDT protein. In particular, we will focus on advanced techniques that can reveal single-stranded and doublestranded DNA gaps and their repair at the single-cell level as well as monitor the progression of single replication forks, such as the specific versions of the DNA fiber and comet assays. This collection of methods may serve as a powerful molecular toolkit to monitor the metabolism of gaps, detect the contribution of relevant pathways and molecular players, as well as characterize the effectiveness of potential inhibitors.
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关键词
RAD18,Postreplication repair,DNA damage tolerance/bypass,Comet assay,DNA fiber
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