Abstract
When a replicative DNA polymerase stalls upon encountering a photoproduct on the template strand, it is relieved by other low-processivity polymerase(s), which insert nucleotide(s) opposite the lesion. Using an alkaline sucrose density gradient sedimentation technique, we previously classified this process termed UV-induced translesion replication (UV-TLS) into two types. In human cancer cells or xeroderma pigmentosum variant (XP-V) cells, UV-TLS was inhibited by caffeine or proteasome inhibitors. However, in normal human cells, the process was insensitive to these reagents. Reportedly, in yeast or mammalian cells, REV3 protein (a catalytic subunit of DNA polymerase ζ) is predominantly involved in the former type of TLS. Here, we studied UV-TLS in fibroblasts derived from the Rev3-knockout mouse embryo (Rev3KO-MEF). In the wild-type MEF, UV-TLS was slow (similar to that of human cancer cells or XP-V cells), and was abolished by caffeine or MG-262. In 2 cell lines of Rev3KO-MEF (Rev3−/− p53−/−), UV-TLS was not observed. In p53KO-MEF, which is a strict control for Rev3KO-MEF, the UV-TLS response was similar to that of the wild-type. Introduction of the Rev3 expression plasmid into Rev3KO-MEF restored the UV-TLS response in selected stable transformants. In some transformants, viability to UV was the same as that in the wild-type, and the death rate was increased by caffeine. Our findings indicate that REV3 is predominantly involved in UV-TLS in mouse cells, and that the REV3 translesion pathway is suppressed by caffeine or proteasome inhibitors.
Highlights
At least five mammalian DNA polymerases are suggested to be implicated in UV-induced translesion replication (TLS): Pols η, ι, ζ, κ and REV1
Our findings indicate that REV3 is predominantly involved in UV-induced translesion replication (UV-TLS) in mouse cells, and that the REV3 translesion pathway is suppressed by caffeine or proteasome inhibitors
The addition of 5 μM MG-262 or 5 mM caffeine to the chase medium significantly delayed conversion in UV-irradiated 1G or 2F cells (Figure 4: 1G and 2F—compare lines 4 and 5 with line 2 or line 3). These results indicate that the expression of exogenous Rev3 in Rev3KO-MEFs restores the UV-TLS response, and that this response is sensitive to proteasome inhibitors and caffeine
Summary
At least five mammalian DNA polymerases are suggested to be implicated in UV-induced translesion replication (TLS) (reviewed in [1]): Pols η, ι, ζ, κ and REV1. Polη and Polζ are markedly involved in UV-TLS in human [4] or vertebrate cells [5]. Using a modified ASDG technique [28], we previously detected the conversion of pulse-labeled replication products in UV-irradiated fibroblasts derived from XP-V patients, showing that UV-TLS is delayed in the cells, but not completely abolished [29]. Unlike in Rev1-deficient MEFs, there was no delay in fork progression after UV irradiation (results from DNA combing assay). We investigated the effects of caffeine and proteasome inhibitors on UV-TLS, and the kinetics of progression for the nascent strand in UV-irradiated Rev3−/− p53−/− MEFs
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.