Motifs of the C-terminal domain of MCM9 direct localization to sites of mitomycin-C damage for RAD51 recruitment

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.

A novel multi-functional role for the MCM8/9 helicase complex in maintaining fork integrity during replication stress

“…Previously, several laboratories including ours have shown that MCM8/9 form nuclear foci, upon damage, primarily from DNA crosslinking agents or after direct DSBs induced from ionizing radiation. 24,30,31 This implicates MCM8/9 in HR, but there is limited information investigating a possible role during fork progression/stalling. To directly examine whether MCM8/9 are involved in maintaining genomic instability during replication stress, a C-terminal GFP-tagged MCM9 fusion construct was transfected into WT 293T cells, after which cells were treated with 2 mM hydroxyurea (HU) for 4 hours, and MCM9 foci formation was monitored ( Fig.…”

“…7a ) that interacted with and recruited RAD51 to sites of MMC induced DNA damage. 30 Therefore, we sought to investigate the role of this MCM9-BRCv motif in maintaining fork stability after HU treatment using DNA fiber analysis. Interestingly, in the absence of HU, fork stability is restored in 9 KO cells when MCM9(BRCv - ) is transfected, implying that the MCM8/9 complex on its own provides some stabilizing context to active replisomes, possibly through its helicase activity ( Fig.…”

“…An interaction with RAD51 through the MCM9 BRCv motif, in particular, is influential in this dynamic protection process and likely stabilizes a subset of stalled forks that are not significantly reversed. 30 Even transfection of a catalytically inactive MCM9 stabilizes forks to a greater level than that of a BRCv - mutant during HU stalling, highlighting the importance of the BRCv motif to recruit Rad51 over that of the any associated ATPase activity utilized for fork progression. MCM8/9 antagonizes the effects of BRCA1 localization, which itself acts to stabilize stalled and reversed forks slightly further downstream to aid in their restart.…”

“…8 KO and 9 KO in parental 293T cells were created using CRISPR/Cas9 technology and confirmed knockout by DNA sequencing and mitomycin C (MMC) sensitivity assays. 30 All cells were cultured at 37 °C with 5% CO 2 in Dulbecco’s modified eagle’s medium (DMEM) (Corning Cellgro) supplemented with fetal bovine serum (FBS) (Atlanta Biologicals) at a 10% working concentration. Plasmid transfections (pEGFPc2-MCM8, pEGFPc2-MCM9, pEGFPc2-MCM9(FR687/8AA) were carried out using LPEI (ThermoFisher) as described.…”

“…Plasmid transfections (pEGFPc2-MCM8, pEGFPc2-MCM9, pEGFPc2-MCM9(FR687/8AA) were carried out using LPEI (ThermoFisher) as described. 30 The MCM8 or MCM9 gene was cloned into the pIRES2-EGFP using traditional restriction site cloning, BglII/BamHI and XhoI/XmaI , respectively. pEGFPc2-MCM9(K358A) was created by a modified QuikChange protocol and screened with a novel inserted restriction enzyme, SmaI (NENB).…”