Understanding the mechanis of mammalian damage tolerance proteins in suppressing damage-specific mutagenesis
- Post-replication repair (PRR) pathways play important roles in restarting stalled replication forks and regulating mutagenesis. In yeast, Rad5-mediated damage avoidance and Rad18-mediated translesion synthesis (TLS) are two forms of PRR. Two Rad5-related proteins, SHPRH and HLTF, have been identified in mammalian cells, but their specific roles in PRR are unclear. Here, we show that HLTF and SHPRH suppress mutagenesis in a damage-specific manner, preventing mutations induced by UV and MMS, respectively. Following UV, HLTF enhances PCNA monoubiquitination and recruitment of TLS polymerase eta, while also inhibiting SHPRH function. In contrast, MMS promotes the degradation of HLTF and the interactions of SHPRH with Rad18 and polymerase kappa. Our data not only suggest that cells differentially utilize HLTF and SHPRH for different forms of DNA damage, but also, surprisingly, that HLTF and SHPRH may coordinate the two main branches of PRR to choose the proper bypass mechanism for minimizing mutagenesis. Though the degradation of HLTF is required for the SHPRH-Rad18 interaction in MMS-damaged cells, knockdown of HLTF is not sufficient to induce SHPRH-Rad18 binding in undamaged cells. Thus, an unidentified factor, in addition to HLTF degradation, is required to fully activate SHPRH and Rad18 upon MMS damage. We have noticed that Rad18 is deubiquitinated after MMS treatment, and that this de-ubiquitination is correlated with the degree of Rad18-SHPRH interaction. Moreover, we show that promoting the ubiquitination of Rad18 has an inverse effect on the interaction with SHPRH both in vitro and in cells. Surprisingly, though, the ubiquitinated Rad18 shows a stronger self-interaction than the wild-type Rad18, opposite from the SHPRH binding results. As the zinc-finger motif of Rad18 has been previously shown to bind ubiquitin, this may be a logical mechanism to control the switch between Rad18 dimerization and its interaction with other proteins. Interestingly, ubiquitinated Rad18 appears to be inactive, not only from loss of its ability to interact with SHPRH, but also through the inability to form damage foci and suppress MMS-induced mutagenesis. Altogether, our data reveal a unique regulation of Rad18 through self-ubiquitination and dimerization. In summary, our studies have revealed a number of new mechanistic insights into the post-replication repair pathways in human cells, including the interesting conclusion that protein degradation, deubiquitination, and other key cell processes may be controlled in a DNA damage-specific manner.
|Type of resource
|electronic; electronic resource; remote
|1 online resource.
|Stanford University, Department of Chemical and Systems Biology.
|Kopito, Ron Rieger
|Kopito, Ron Rieger
|Statement of responsibility
|Submitted to the Department of Chemical and Systems Biology.
|Ph.D. Stanford University 2012
- © 2012 by Jia-Ren Lin
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