Genetic and physical interactions between Pol\u03b7 and Rev1 in response to UV-induced DNA damage in mammalian cells

Tonghui Bi,Xiaohong Niu,Wei Xiao,Chunping Qin

doi:10.1038/s41598-021-00878-3

Tonghui Bi, Xiaohong Niu + Show 2 more

Open Access

https://doi.org/10.1038/s41598-021-00878-3

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Abstract

In response to UV irradiation, translesion DNA synthesis (TLS) utilizes specialized DNA polymerases to bypass replication-blocking lesions. In a well-established polymerase switch model, Polη is thought to be a preferred TLS polymerase to insert correct nucleotides across from the thymine dimer, and Rev1 plays a scaffold role through physical interaction with Polη and the Rev7 subunit of Polζ for continual DNA synthesis. Defective Polη causes a variant form of xeroderma pigmentosum (XPV), a disease with predisposition to sunlight-induced skin cancer. Previous studies revealed that expression of Rev1 alone is sufficient to confer enhanced UV damage tolerance in mammalian cells, which depends on its physical interaction with Polζ but is independent of Polη, a conclusion that appears to contradict current literature on the critical roles of Polη in TLS. To test a hypothesis that the Rev1 catalytic activity is required to backup Polη in TLS, we found that the Rev1 polymerase-dead mutation is synergistic with either Polη mutation or the Polη-interaction mutation in response to UV-induced DNA damage. On the other hand, functional complementation of polH cells by Polη relies on its physical interaction with Rev1. Hence, our studies reveal critical interactions between Rev1 and Polη in response to UV damage.

Highlights

In response to UV irradiation, translesion DNA synthesis (TLS) utilizes specialized DNA polymerases to bypass replication-blocking lesions
We previously reported that UV damage tolerance mediated by PCNA-Ub fusion is dependent on Rev[1] but independent of Polη[37]
By using an RPA nuclear focus formation assay as an indication of TLS activity after UV irradiation[37,40], it was found that Rev1-L1170A and Rev1-V1188A protected cells to a level comparable to that of Rev[1], while C-terminal domain (CTD)-Y1242A and CTD-L1246A lost Rev1 functions[36] (Supplementary Fig. S1), which indicates that DNA damage tolerance (DDT) provided by Rev[1] does not require its physical interaction with Polη

Summary

Introduction

In response to UV irradiation, translesion DNA synthesis (TLS) utilizes specialized DNA polymerases to bypass replication-blocking lesions. Previous studies revealed that expression of Rev[1] alone is sufficient to confer enhanced UV damage tolerance in mammalian cells, which depends on its physical interaction with Polζ but is independent of Polη, a conclusion that appears to contradict current literature on the critical roles of Polη in TLS. It has been reported that Polη is required for the recruitment of Rev[1] to the damage site through its RIR motifs, but ectopic expression of the RIR-defective Polη does not affect its ability to protect cells from UV-induced killing and m utagenesis[17]. The current study further addressed genetic and physical interactions between Rev[1] and Polη, which allowed us to conclude that the Rev[1] polymerase can play a backup role in the absence of Polη and that Polη requires its RIR motifs to protect cells from UV-induced DNA damage

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