Abstract
Translesion DNA synthesis (TLS) mediated by low-fidelity DNA polymerases is an essential cellular mechanism for bypassing DNA lesions that obstruct DNA replication progression. However, the access of TLS polymerases to the replication machinery must be kept tightly in check to avoid excessive mutagenesis. Recruitment of DNA polymerase η (Pol η) and other Y-family TLS polymerases to damaged DNA relies on proliferating cell nuclear antigen (PCNA) monoubiquitylation and is regulated at several levels. Using a microscopy-based RNAi screen, here we identified an important role of the SUMO modification pathway in limiting Pol η interactions with DNA damage sites in human cells. We found that Pol η undergoes DNA damage- and protein inhibitor of activated STAT 1 (PIAS1)-dependent polySUMOylation upon its association with monoubiquitylated PCNA, rendering it susceptible to extraction from DNA damage sites by SUMO-targeted ubiquitin ligase (STUbL) activity. Using proteomic profiling, we demonstrate that Pol η is targeted for multisite SUMOylation, and that collectively these SUMO modifications are essential for PIAS1- and STUbL-mediated displacement of Pol η from DNA damage sites. These findings suggest that a SUMO-driven feedback inhibition mechanism is an intrinsic feature of TLS-mediated lesion bypass functioning to curtail the interaction of Pol η with PCNA at damaged DNA to prevent harmful mutagenesis.
Highlights
Translesion DNA synthesis (TLS) mediated by low-fidelity DNA polymerases is an essential cellular mechanism for bypassing DNA lesions that obstruct DNA replication progression
SiRNA-transfected cells were treated with cisplatin to induce DNA damage, and acquisition and quantification of the resulting GFP-Pol foci were performed using quantitative imagebased cytometry (QIBC) [42], allowing for the ranking of all siRNAs according to their impact on GFP-Pol foci counts (Fig. 1, A and B; Table S1)
Based on an unbiased screen for post-translational modification (PTM)-mediated regulators of Pol interaction with DNA damage sites, we here provide evidence that one cellular mechanism for limiting Pol -mediated TLS involves a built-in feedback inhibition circuit driven by SUMOylation
Summary
Translesion DNA synthesis (TLS) mediated by low-fidelity DNA polymerases is an essential cellular mechanism for bypassing DNA lesions that obstruct DNA replication progression. Genome duplication generally proceeds in a highly processive and accurate manner, due to the extremely high fidelity and proofreading activity of normal, replicative DNA polymerases [3] Because these polymerases are unable to accommodate most damaged or modified DNA structures in their active sites, the occurrence of DNA lesions in S phase may obstruct the progression of the advancing replication machinery. Because TLS polymerases have flexible active sites and lack proofreading activity, their fidelities are orders of magnitude lower than those of replicative DNA polymerases, TLS is much more error-prone than normal DNA replication [5] Due to these inherent properties, TLS plays a double-edged role in genome stability maintenance: on the one hand, the bypass of DNA lesions during replication by damage-tolerant polymerases provides cells with the flexibility needed to avoid the potentially much more severe consequences of collapsed replication forks. TLS must be kept tightly in check at all times, and multiple regulatory mechanisms have evolved to enable a carefully balanced level of TLS that allows DNA lesion bypass while suppressing excessive mutagenesis
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