Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5′ flap DNA: basis of interstrand cross-link repair by FAN1

Abstract

Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of crosslinks, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI-FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 59 flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domain playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 59 flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.

Fan1 deficiency results in DNA interstrand cross-link repair defects, enhanced tissue karyomegaly, and organ dysfunction

“…Recent crystallographic data revealed that the SAP domain interacts extensively with the DNA (Gwon et al 2014;Wang et al 2014;Zhang and Walter 2014), suggesting that direct DNA binding to the ICL might be responsible for the recruitment of FAN1 to sites of DNA damage. To investigate the contribution of the UBZ and the SAP domains to the localization of FAN1 at ICLs, we studied the recruitment of human GFP-tagged FAN1 (GFP-hFAN1) to psoralen-induced ICLs in U2OS cells ( Fig.…”

FANCD2-associated Nuclease 1, but Not Exonuclease 1 or Flap Endonuclease 1, Is Able to Unhook DNA Interstrand Cross-links in Vitro

“…1A) with T4 polynucleotide kinase (New England Biolabs) and [␥- 32 P]ATP and annealing with oligonucleotide III as described above. The 3Ј-labeled cross-linked substrates (Figs.…”

Structural mechanism of DNA interstrand cross-link unhooking by the bacterial FAN1 nuclease

“…However, a number of studies suggested that the cross-link repair function of FAN1 is not within the FA pathway as: (i) homologs of FAN1 have been found in bacteria, archaea, and unicellular eukaryotes which lack FA proteins (19,(21)(22)(23), (ii) mutations in FAN1 do not cause FA, but instead a kidney disorder called karyomegalic interstitial nephritis (KIN) (24), (iii) N-terminal UBZ domain is dispensable for ICL repair, but is required for genomic maintenance, suggesting that the interaction of FAN1 with ID serves a function outside of ICL repair (25)(26)(27). Nevertheless, the sensitivity of FAN1-deficient cells to ICL-forming agents implicates FAN1 in ICL resolution independently of the FA pathway.…”

“…FAN1 incises substrates 2-5 nucleotides into the duplex from the ss/ds junction of DNA with a 5' flap or a nick, followed by 5' to 3' exonucleolytic cleavage of the duplex even in presence of an ICL (17)(18)(19)(20)23,(32)(33)(34).…”

“…Recently, several crystal structures of FAN1 bound to DNA have been reported and mechanisms of how they degrade ICL-containing DNA substrates have been proposed (23,(33)(34)(35). To date, these are the only structures of an ICL-repair nuclease bound to DNA.…”