The ubiquitin-binding domain of DNA polymerase η directly binds to DNA clamp PCNA and regulates translesion DNA synthesis

Kodavati Manohar,Prashant Khandagale,Shraddheya Kumar Patel,Jugal Kishor Sahu,Narottam Acharya

doi:10.1016/j.jbc.2021.101506

Kodavati Manohar, Prashant Khandagale + Show 3 more

Open Access

https://doi.org/10.1016/j.jbc.2021.101506

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Abstract

DNA polymerase eta (Polη) is a unique translesion DNA synthesis (TLS) enzyme required for the error-free bypass of ultraviolet ray (UV)-induced cyclobutane pyrimidine dimers in DNA. Therefore, its deficiency confers cellular sensitivity to UV radiation and an increased rate of UV-induced mutagenesis. Polη possesses a ubiquitin-binding zinc finger (ubz) domain and a PCNA-interacting-protein (pip) motif in the carboxy-terminal region. The role of the Polη pip motif in PCNA interaction required for DNA polymerase recruitment to the stalled replication fork has been demonstrated in earlier studies; however, the function of the ubz domain remains divisive. As per the current notion, the ubz domain of Polη binds to the ubiquitin moiety of the ubiquitinated PCNA, but such interaction is found to be nonessential for Polη's function. In this study, through amino acid sequence alignments, we identify three classes of Polη among different species based on the presence or absence of pip motif or ubz domain and using comprehensive mutational analyses, we show that the ubz domain of Polη, which intrinsically lacks the pip motif directly binds to the interdomain connecting loop (IDCL) of PCNA and regulates Polη's TLS activity. We further propose two distinct modes of PCNA interaction mediated either by pip motif or ubz domain in various Polη homologs. When the pip motif or ubz domain of a given Polη binds to the IDCL of PCNA, such interaction becomes essential, whereas the binding of ubz domain to PCNA through ubiquitin is dispensable for Polη's function.

Highlights

Y-family DNA polymerases (Pols) are a group of non-essential enzymes that play an imperative role during translesion DNA synthesis (TLS) [1,2]
As PCNA gets monoubiquitinated via Rad6-Rad18 ubiquitination conjugating system during TLS [13,14,15,16], it was proposed that the TLS polymerases bind to the ubiquitin moiety attached on PCNA through UBDs, and UBD-Ub-PCNA interaction is indispensable for the recruitment of TLS pols to PCNA
We identified a group of polymerase eta (Pol) s only from fungi belonging to category III that seem to lack a pip motif at their usual location intrinsically

Summary

Introduction

Y-family DNA polymerases (Pols) are a group of non-essential enzymes that play an imperative role during translesion DNA synthesis (TLS) [1,2]. In addition to the pip motif, several highly conserved ubiquitin (Ub) binding domains (UBDs) have been identified in the C-terminal regions of Y-family pols, which regulate their TLS function [12]. The C-terminal amino acid sequence of Pol s showed maximum variations, Journal Pre-proof and some of the members lacked either pip motif or ubz domain (Table 1, Fig. S1). They were classified into three distinct categories based on the presence or absence of pip motif and ubz domain. Considering previous reports and the current study, we suggest two modes of PCNA interaction by Pol s from various organisms

Results

Competing Interest Statement

22 SpRad30