Abstract
The MCM8/9 complex is implicated in aiding fork progression and facilitating homologous recombination (HR) in response to several DNA damage agents. MCM9 itself is an outlier within the MCM family containing a long C-terminal extension (CTE) comprising 42% of the total length, but with no known functional components and high predicted disorder. In this report, we identify and characterize two unique motifs within the primarily unstructured CTE that are required for localization of MCM8/9 to sites of mitomycin C (MMC)-induced DNA damage. First, an unconventional “bipartite-like” nuclear localization (NLS) motif consisting of two positively charged amino acid stretches separated by a long intervening sequence is required for the nuclear import of both MCM8 and MCM9. Second, a variant of the BRC motif (BRCv) similar to that found in other HR helicases is necessary for localization to sites of MMC damage. The MCM9-BRCv directly interacts with and recruits RAD51 downstream to MMC-induced damage to aid in DNA repair. Patient lymphocytes devoid of functional MCM9 and discrete MCM9 knockout cells have a significantly impaired ability to form RAD51 foci after MMC treatment. Therefore, the disordered CTE in MCM9 is functionally important in promoting MCM8/9 activity and in recruiting downstream interactors; thus, requiring full-length MCM9 for proper DNA repair.
Highlights
Helicases contribute to several steps in the recombination pathways either facilitating or dissolving hybrid DNA recombinants [5]
Like that found for breast cancer type 1 susceptibility protein (BRCA1)/2-deficient cells, MCM8 or 9 deficient cells are hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors indicating a link between fork progression and BRAC1/2-mediated homologous recombination (HR) repair that can be exploited in the clinic for synthetically lethal therapies with platinum-based DNA cross-linking agents [32]
As we had previously characterized MCM8 and MCM9 mutations of premature ovarian failure (POF) patients within consanguineous families, we examined whether these conditioned mutations in MCM9 impaired RAD51 foci formation with mitomycin C (MMC) treatment (Fig. 7, A and B and Fig. S7). 8AIV-3 designates patient Epstein– Barr virus (EBV) transformed lymphocytes from an affected MCM8 family [17] but is fully WT for both MCM8 and MCM9, acts as a suitable control, and shows significant RAD51 foci after MMC damage as expected. 9BII-4 designates lymphocytes from a heterozygous family member with one WT allele and one splice site mutation that eliminates the C-terminal extension (CTE) of MCM9 [18]
Summary
We and others have shown previously that MCM8 and MCM9 are generally localized to the nucleus and form nuclear foci after damage with MMC [11, 17]. As MCM9Cterm had predominantly nuclear staining but MCM9M did not, we searched for nuclear localization sequences (NLS) in the CTE and found four conserved, highconfidence putative NLS sequences (pNLS) (Fig. S3) These four pNLS sequences were individually mutated in MCM9LGFP, transfected into U2OS cells, and the localization was examined by confocal microscopy (Fig. 3, A and B). Mutation of either NLS1 or NLS2 shows a significant decrease in the N/C ratio This result confirms a requirement for both NLS1 and NLS2 in the CTE of MCM9 for efficient import into the nucleus. Transfection of MCM9L-GFP or MCM9M-GPF into MCM8KO cells (Fig. 4C) shows essentially the same results (as, B and C) with nuclear and cytoplasmic staining, respectively. MCM8 is imported into the nucleus complexed with MCM9 as directed by the “bipartite-like” NLS1/NLS2
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