FAN1

FANCD2/FANCI-associated nuclease 1 (KIAA1018) is an is_associated_with::enzyme that in is_associated_with::humans is encoded by the FAN1 is_associated_with::gene. It is a structure dependent endonuclease and a member of the is_associated_with::myotubularin-related class 1 cysteine-based is_associated_with::protein is_associated_with::tyrosine is_associated_with::phosphatases. It is thought to play an important role in the is_associated_with::Fanconi Anemia (FA) pathway.

Structure


FAN1 is a protein of 1017 is_associated_with::amino acids. Several crystal structures of the residues 373-1017 have been characterized. This portion of FAN1 contains three domains: an SAP domain (primary-DNA binding domain), a TPR domain (mediating interdomain interaction and dimerization interface) and the is_associated_with::virus-type replication-repair nuclease module (VRR_NUC, catalytic cite) (Figure 1). is_associated_with::DNA binding promotes dimerization of FAN1 in a "head to tail" fashion.

The SAP region contains three major components: α9, α5β1, and α7. The core helix α9 stabilizes the protein as it moves through dimer configurations and mediates the interactions between α5β1 and α7 as they adjust their positions. These three configurations are the substrate scanning, substrate latching and substrate unwinding forms (figure 2).

In the FAN1 dimer, the SAP regions of both FAN1 enzymes make contact with the DNA duplex (dsDNA). This double contact facilitates DNA induced dimerization, as well as guiding the single stranded (ssDNA) into the SAP domain of the downstream enzyme (PSAP). The SAP domain of the upstream FAN1 component enzyme (ASAP) aids in guiding the DNA to PSAP.

The SAP surface facing the is_associated_with::catalytic site is the most conserved region between FAN1 homologs. It is positively charged for favorable hydrogen bonding and electrostatic interactions with DNA. In particular, residues Y374 and Y436 form hydrogen bonds with the phosphate backbone. FAN1 can bind DNA in either direction. However, when the 5' flab is facing away from the VRR_NUC site, substrate latching and unwinding cannot occur. The unresolved portion of FAN1 contains a Zinc finger at the N terminus called a UBZ region. This is present in proteins that bind to is_associated_with::ubiquitinated proteins, and is highly conserved across is_associated_with::eukaryotes. This Zinc finger is crucial for recruitment to the ubiquitinated FANCD2/FANCI complex, and is found in other nucleases. The VRR_Nuc catalytic domain is located at the C terminus and contains the endonuclease functionality. FAN1 is the first known instance of a virus type replication-repair nuclease module in is_associated_with::eukaryotes. It is normally found as a standalone domain in bacterial and viral Holliday Junction Resolvases (HJR). FAN1 does not exhibit any activity on Holliday Junction (HJ) substrates. A subdomain of SAP consisting of six is_associated_with::α helices connected to the VRR_Nuc region is thought to inhibit HJR activity.

Function


Repair of interstrand is_associated_with::DNA crosslinks is triggered when the DNA replication fork is unable to continue. The FA proteins play an elaborate role with FAN1 to remove these ICLs. The pathway consists of 15 known proteins. Three of them form the FA AP24-MHF1/2 complex which recognizes the ICL (from stalled replication forks). This recruits the FA core complex, which consists of 8 proteins. This complex monoubiquitinates FANCD2 and FANCI, which allows it to form a heterodimer. It is this complex that recruits FAN1 as well as other nucleases such as is_associated_with::SLX4. FAN1 is typically localized in the nucleus, but forms very distinct loci at damaged regions when ICLs are present.

The FAN1 protein possesses is_associated_with::endonuclease and is_associated_with::exonuclease functions to remove ICLs. It is thought that this process consists of unhooking the crosslink and separating the DNA strands through two incision events, yielding one strand with a crosslinked nucleotide and another strand with a gap. FAN1 preferentially acts as a 5’ flap endonuclease. This is illustrated in Figure 2, which shows the sequence of substrate scanning, latching, and unwinding. It usually cleaves about 5 nucleotides from a junction. FAN1 will also incise at splayed arms, three way junctions, and 3’ flaps (in order of decreasing preference). In high concentrations FAN1 has been shown to exhibit 3’ 5’ exonuclease activity. In blunt end substrates, FAN1 has also 5’ recessed ends. However, FAN1 does not appear to bind to single stranded DNA.

The presence of the FANCD2/FANCI complex is unaffected by knockdown of FAN1. This is because FAN1 acts downstream to the recruitment of FANCD2/FANCI. FAN1 has also been shown to increase the frequency of is_associated_with::homologous recombination. This suggests that the gapped intermediate that forms following ICL unhooking may be repaired through HR when homologous chromosomes are present. FAN1 does not appear to be involved in other types of is_associated_with::DNA repair, as it does not localize to DNA upon is_associated_with::irradiation.

Clinical significance
Mutations affecting the function of the 15 known FA genes are associated with Fanconi anemia, a recessive autosomal disorder. It is characterized by congenital abnormalities as well as is_associated_with::anemia, is_associated_with::bone marrow failure, and is_associated_with::cancer predisposition in childhood. However, some patients have “unassigned” Fanconi Anemia where no is_associated_with::mutations in the known FA genes can be found. Mutations in FAN1 can result in chronic is_associated_with::kidney diseases and neurological conditions such as is_associated_with::schizophrenia. However, recent research has called into question the categorization of FAN1 as an FA gene. In 2015 researchers studied four individuals with chromosomal is_associated_with::microdeletion of 15q13.3. Analysis of is_associated_with::blood samples revealed only mild ICL agent sensitivity and chromosomal fragility consistent with Fanconi Anemia.

A deficiency of FAN1 increases in vitro sensitivity to cisplatin and mitomycin C, two crosslinking agents FAN1 is also able to repair mitomycin C induced double strand breaks.