FANCD2-Associated Nuclease 1 Partially Compensates for the Lack of Exonuclease 1 in Mismatch Repair

Katja Kratz,Saho Kobayashi-Era,Shunichi Takeda,Josef Jiricny,Xueqing Zou,Joanna I Loizou,Andreia Oliveira,Serena Nik-Zainal,Gene Koh,Hiroyuki Sasanuma,Alessandro A Sartori,Mariela Artola-Borán,Masataka Tsuda,Julia Richter,Goncalo Oliveira,Shunsuke Kobayashi

doi:10.1128/mcb.00303-21

Abstract

ABSTRACTGermline mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1, and PMS2 are linked to cancer of the colon and other organs, characterized by microsatellite instability and a large increase in mutation frequency. Unexpectedly, mutations in EXO1, encoding the only exonuclease genetically implicated in MMR, are not linked to familial cancer and cause a substantially weaker mutator phenotype. This difference could be explained if eukaryotic cells possessed additional exonucleases redundant with EXO1. Analysis of the MLH1 interactome identified FANCD2-associated nuclease 1 (FAN1), a novel enzyme with biochemical properties resembling EXO1. We now show that FAN1 efficiently substitutes for EXO1 in MMR assays and that this functional complementation is modulated by its interaction with MLH1. FAN1 also contributes to MMR in vivo; cells lacking both EXO1 and FAN1 have an MMR defect and display resistance to N-methyl-N-nitrosourea (MNU) and 6-thioguanine (TG). Moreover, FAN1 loss amplifies the mutational profile of EXO1-deficient cells, suggesting that the two nucleases act redundantly in the same antimutagenic pathway. However, the increased drug resistance and mutator phenotype of FAN1/EXO1-deficient cells are less prominent than those seen in cells lacking MSH6 or MLH1. Eukaryotic cells thus apparently possess additional mechanisms that compensate for the loss of EXO1.

Highlights

Germline mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1, and PMS2 are linked to cancer of the colon and other organs, characterized by microsatellite instability and a large increase in mutation frequency
Having discovered FANCD2-associated nuclease 1 (FAN1)/KIAA1018 as an interactor of MLH1 [23] and having shown that it is a 59-flap endonuclease and a 59-39 exonuclease [26, 30], we hypothesized that the protein might play a role in MMR as a backup for EXO1, which has similar activities and which interacts with MLH1 [31,32,33]
We considered the possibility that the FAN1/MLH1 complex is required to be posttranslationally modified during S-phase in order to be active

Summary

Introduction

Germline mutations in the mismatch repair (MMR) genes MSH2, MSH6, MLH1, and PMS2 are linked to cancer of the colon and other organs, characterized by microsatellite instability and a large increase in mutation frequency. Compared to MMR-proficient cells, up to 100-fold higher rates of substitution mutations and high MSI are associated with loss of MSH2, MLH1, or PMS2, whereas inactivation of MSH6 causes a similar increase in substitution mutations, but MSI is limited largely to mononucleotide repeats This is due to the partial functional redundancy between MSH6 and MSH3 in the mismatch recognition factors MutSa (MSH2/MSH6) [4,5,6] and MutSb (MSH2/MSH3) [7]. The above-described mechanism has been deduced from a large number of biochemical investigations that made use of a nicked mismatch-containing plasmid substrate (similar to that shown in Fig. 1A) and extracts of MMR-proficient or -deficient cells [19, 20] These efforts culminated in the reconstitution of the minimal human MMR system consisting of MutSa, MutLa, RFC, PCNA, replication protein A (RPA), DNA polymerase d , EXO1, ATP, and deoxynucleoside triphosphate (dNTPs) [21, 22]

Methods

Results

Conclusion