McDonald lab Signalling and Structural Biology Laboratory
: Structural biology of the XPF/MUS81/FANCM endonuclease family
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Mammalian endonucleases play key roles in several DNA repair pathways including nucleotide excision repair (NER), interstrand crosslink repair (ICL), recombination and replication fork repair, and thereby help maintain genomic integrity.
Figure 2A: Identification of a DNA-binding winged helix domain within Mus81. Previous studies had missed the presence of this domain within Mus81. This structure was determined by NMR. B. Crystal structure of a C-terminal fragment of FANCM complexed to its FAAP24 partner protein. The FANCM (HhH)2 hairpins are buried whilst FAAP24 (HhH)2 domain binds dsDNA. The trajectory of the dsDNA relative to the FANCM pseudo-nuclease domain is distinct from our previous archeal XPF-dsDNA structure. For dsDNA to engage both the FAAP24 (HhH)2 hairpins and the FANCM pseudo-nuclease domain it must be bent from a linear B-DNA conformation.
We have been studying the XPF family of structure-specific endonucleases as potential translational research projects. Our rationale is that understanding endonuclease catalytic mechanism and identifying a chemical inhibitor that could block NER and ICL, may render a wider range of tumour cells sensitive to DNA-damaging agents.
The human XPF family comprises at least three active hetero-dimeric endonuclease complexes; XPF-ERCC1, MUS81-EME1 and the largely uncharacterised MUS81-EME2 complex. A fourth complex, FANCM-FAAP24, belongs to the XPF family but does not exhibit endonuclease activity.
This year we have reported the identification and structure of a DNA-binding winged helix domain within human MUS81 that had been missed in previous studies (Fadden et al., 2013; Nucleic Acids Res. 41(21): 9741-52).
We show that this domain can bind dsDNA and plays a role in positioning the scissile bond within junction substrates (Figure 2A).
We also recently reported the crystal structure of a C-terminal fragment of FANCM complexed to its FAAP24 partner protein and a short dsDNA oligonucleotide (Coulthard et al., 2013; Structure. 21: 1648-58). In this structure, the FANCM (HhH)2 hairpins are buried within an interface with the FANCM pseudo-nuclease domain.
The dsDNA trajectory is distinct from our previous archeal XPF-dsDNA structure suggesting that in order to engage both the FAAP24 (HhH)2 hairpins and the FANCM pseudo-nuclease domain it must deviate substantially from linear B-DNA.
Collaborations with the Genetic Recombination and Architecture and Dynamics of Macromolecular Machines Groupsat the Clare Hall Laboratories have validated functionally important roles for the FAAP24 (HhH)2 and FANCM pseudo-nuclease domain consistent with our structure and led to an EM structure using full length FANCM-FAAP24 complex.
We are also continuing to work collaboratively with CRT to discover chemical inhibitors of human XPF-ERCC1 using our published fluorescence-based assay (Bowles et al., 2012; Nucleic Acids Res. 40: e101).
