FANCM is involved in eukaryotic DNA-damage recognition and activates the Fanconi anemia (FA) pathway through complex formation. MHF is one of the FANCM-associating components and contains a histone-fold DNA-binding domain. Loss of the FANCM-MHF interaction compromises the activation of the FA pathway, resulting in chromosomal instability. Thus, formation of the FANCM-MHF complex is important for function, but its nature largely remains elusive. Here, the aim was to reveal the molecular and structural basis for the stability of the FANCM-MHF complex. A recombinant tripartite complex containing chicken FANCM (MHF interaction region), MHF1 and MHF2 was expressed and purified. The purified tripartite complex was crystallized under various conditions and three different crystals were obtained from similar crystallization conditions. Unexpectedly, structure determination revealed that one of the crystals contained the FANCM-MHF complex but that the other two contained the MHF complex without FANCM. How FANCM dissociates from MHF was further investigated and it was found that the presence of 2-methyl-2,4-pentanediol (MPD) and an oxidative environment may have promoted its release. However, under these conditions MHF retained its complexed form. FANCM-MHF interaction involves a mixture of hydrophobic/hydrophilic interactions, and chicken FANCM contains several nonconserved cysteines within this region which may lead to aggregation with other FANCM-MHF molecules. These results indicate an unexpected nature of the FANCM-MHF complex and the data can be used to improve the stability of the complex for biochemical and structural analyses.
Keywords: DNA binding; DNA repair; X-ray crystallography; histone fold; protein complex.
open access.