Importance of homo-dimerization of Fanconi-associated nuclease 1 in DNA flap cleavage

Abstract

Fanconi-associated nuclease 1 (FAN1) removes interstrand DNA crosslinks (ICLs) through its DNA flap endonuclease and exonuclease activities. Crystal structures of human and bacterial FAN1 bound to a DNA flap have been solved. The Pseudomonas aeruginosa bacterial FAN1 and human FAN1 (hFAN1) missing a flexible loop are monomeric, while intact hFAN1 is homo-dimeric in structure. Importantly, the monomeric and dimeric forms of FAN1 exhibit very different DNA binding modes. Here, we interrogate the functional differences between monomeric and dimeric forms of FAN1 and provide an explanation for the discrepancy in oligomeric state between the two hFAN1 structures. Specifically, we show that the flexible loop in question is needed for hFAN1 dimerization. While monomeric and dimeric bacterial or human FAN1 proteins cleave a short 5' flap strand with similar efficiency, optimal cleavage of a long 5' flap strand is contingent upon protein dimerization. Our study therefore furnishes biochemical evidence for a role of hFAN1 homodimerization in biological processes that involve 5' DNA Flap cleavage.

Exome sequencing of individuals with Huntington’s disease implicates FAN1 nuclease activity in slowing CAG expansion and disease onset

“…Double-stranded DNA substrate containing a 5′ single-strand overhang with an IR-700 fluorescent label (5′ flap DNA) was generated by annealing three oligonucleotides (TOM112, TOM117 and TOM122, based on published sequences 64 ) at 95 °C for 10 minutes and cooling slowly to room temperature overnight. Nuclease assays were carried out by pre-incubating 10 nM Nus-His-FAN1 protein, normalized using FAN1 band intensities from SDS-PAGE (Supplementary Fig.…”

FAN1, a DNA Repair Nuclease, as a Modifier of Repeat Expansion Disorders

“…The FAN1 dimer-DNA crystal structure displays three different DNA binding modes (’substrate-scanning’, ‘substrate-latching’, and ‘substrate-unwinding’), which determine distinct mechanisms for DNA cleavage [ 41 ]. Along this line, FAN1 homodimers process long (40 bp) but not short flap (1-5 bp) DNA substrates [ 42 ]. Besides their role in tethering and orienting the DNA substrate for subsequent cleavage, these homodimers possibly evolved to bind DNA in a conformation that simultaneously allows interactions with PCNA, FANCD2-FANCI and other proteins ( Fig.…”

FAN1 exo- not endo-nuclease pausing on disease-associated slipped-DNA repeats: A mechanism of repeat instability

“…Because the HD-modifying FAN1 p.R507H is proximal to residues 510-518, required for DNA-induced FAN1-FAN1 dimerization (Rao et al, 2018;Wang et al, 2014), we tested the hypothesis that FAN1 variants may alter dimerization upon slipped-DNAs. FAN1 alone is monomeric (Pennell et al, 2014), but DNA binding promotes ''head-to-tail'' FAN1 dimerization and processing (Jin and Cho, 2017;Zhao et al, 2014).…”

“…Because FAN1 dimers differentially process long versus short flaps (Rao et al, 2018), we tested whether FAN1 can dimerize on DNAs with long slipped outs of 20 repeats. All FAN1 forms showed similar abilities to dimerize on slipped-DNA (Figure S3C).…”