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.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- BER
- base excision repair;
- BRC
- breast cancer;
- BRCA1
- breast cancer type 1 susceptibility protein;
- BRCA2
- breast cancer type 2 susceptibility protein;
- BME
- β-mercaptoethanol;
- BRCv
- BRC variant motif;
- BSA
- bovine serum albumin;
- CD
- circular dichroism;
- cNLS
- classic NLS;
- CTE
- C-terminal extension;
- CPT
- cisplatin;
- DAPI
- 4’,6-diamindino-2-phenylindole;
- DMEM
- Dulbecco’s modified Eagle’s medium;
- DSB
- double-strand break;
- dsDNA
- double-stranded DNA;
- EBV
- Epstein-Barr virus;
- EDTA
- ethylenediaminetetraacetic acid;
- FA
- Fanconi anemia;
- FBS
- fetal bovine serum;
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase;
- GFP
- green fluorescent protein;
- GST
- glutathione S-transferase;
- HEK
- human embryonic kidney (cells);
- HEPES
- hydroxyethyl piperazineethanesulfonic acid;
- HR
- homologous recombination;
- HRP
- horseradish peroxidase;
- ICL
- interstrand crosslink;
- IPTG
- isopropyl β-D-thiogalactopyranosidase;
- KD
- knockdown;
- KO
- knockout;
- LPEI
- linear polyethyleneimine;
- MCM
- minichromosomal maintenance;
- MMC
- mitomycin-C;
- MMR
- mismatch repair;
- MRN
- Mre11/ Rad50/ Nbs1 complex;
- NER
- nucleotide excision repair;
- NLS
- nuclear localization signal;
- NTD
- N-terminal domain;
- OD
- ocular density;
- PARP
- poly(ADP-ribose) polymerase;
- PBS
- phosphate-buffered saline;
- pCHK1
- phosphorylated checkpoint kinase 1;
- PMSF
- phenylmethylsulfonyl fluoride;
- pNLS
- putative NLS;
- PVDF
- polyvinylidene difluoride;
- RPA
- replication protein A;
- RPMI
- Roswell Park Memorial Institute media;
- ssDNA
- single-stranded DNA;
- SDS-PAGE
- sodium dodecyl sulfate polyacrylamide gel electrophoresis;
- TEV
- tobacco etch virus;
- TLS
- translesion synthesis;
- U2OS
- human bone osteosarcoma epithelial cells;
- WT
- wild type;
- XRCC
- X-ray repair cross complementing