Student Theses and Dissertations

Date of Award

2023

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Agata Smogorzewska

Keywords

FAN1, DNA interstrand crosslink repair, karyomegalic interstitial nephritis (KIN), SNM1A redundancy, Huntington’s disease, MIP-box–MLH1 interaction

Abstract

Exogenous and endogenous insults to genomic integrity are an existential threat to every species, and therefore robust DNA damage repair mechanisms have evolved to protect against genotoxic threats. FANCD2 and FANCI associated nuclease 1 (FAN1) has roles in protection against two major threats to genomic stability: DNA interstrand crosslinks (ICLs), and the expansion of trinucleotide repeats. ICLs are highly deleterious lesions that disrupt replication, are destructive to dividing cells, and are extremely toxic to the hematopoietic system. The replication-dependent Fanconi anemia (FA) pathway defends against ICL toxicity in rapidly dividing cells. However, while it is critical for protecting the bone marrow, the FA pathway is ineffective in the less-proliferative tissues of the kidney and liver. FAN1 interacts with FA pathway proteins and has been shown to process ICLs in biochemical studies. Depletion of FAN1 sensitizes cells to ICL-inducing agents in vitro, but does not cause bone marrow failure in vivo. Rather, it leads to a hereditable chronic kidney disease called karyomegalic interstitial nephritis (KIN), characterized by widespread karyomegaly, end stage renal disease, and liver dysfunction, suggesting FAN1 may play an important genoprotective role in the kidney and liver. Yet a complete picture of the role of FAN1 in DNA damage repair has been hampered by potential compensatory activity of other nucleases in the setting of FAN1 deficiency. The Smogorzewska laboratory has previously shown evidence of redundancy between FAN1 and the nuclease SNM1A/DCLRE1A, which has also been shown to process ICLs. Here, I describe a synergistic ICL repair deficiency phenotype in mouse embryonic fibroblasts derived from Fan1−/− Snm1a−/− (dKO) mice, which exceeds that of FA pathway-deficient cells. In vivo, dKO mice exhibit profound karyomegaly in the kidney and liver. However, the mice exhibit no spontaneous bone marrow deficiency. Even when the hematopoietic system is chronically stressed with a viral memetic the dKOs exhibit no bone marrow failure. Taken together, my results demonstrate that FAN1 and SNM1A participate together in critical genome maintenance activity where the FA-pathway is insufficient. In addition to driving KIN, variants in FAN1 have also been associated with earlier onset of Huntington’s disease (HD), a trinucleotide repeat expansion disease. Depletion of FAN1 increases CAG repeat expansion rate in vitro and in a mouse model of HD. FAN1 also interacts with the mismatch repair (MMR) protein MLH1, which, together with MLH3, is indispensable for CAG repeat expansion in HD. Here, I show that FAN1 interacts with MLH1 via a well conserved MLH1- interacting peptide motif (MIP-box) on FAN1, which is necessary for FAN1-MLH1 interaction. I identify other human MIP-box proteins, including EXO1 and BLM, using in silico modeling I locate the MIP-box binding site (MIS) on MLH1, and using in vitro experiments I show that mutating the MIS abrogates MLH1 interaction with FAN1, EXO1, and BLM. Using biochemical assays, I show that all three MIP-box proteins bind the MLH1-MLH3 heterodimer directly. Based on in silico models, I predict this direct interaction is between MIP-box proteins and MLH1, and that docking of a MIP-box in the MLH1 MIS does not prevent the formation of the MMR heterodimers MLH1-MLH3, MLH1-PMS2, and MLH1- PMS1. A biomathematical model of competition between MIP-box proteins for binding the MLH1 MIS suggests increased interaction between BLM and MLH1 when FAN1 or EXO1 is depleted. I propose a model in which FAN1 suppresses CAG repeat expansion by regulating the amount of BLM that can bind MLH1, which delivers BLM to CAGinduced hairpins, driving repeat expansion. Finally, I describe the experiments necessary to validate the model.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

License and Reuse Information

Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.

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