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

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100355

1083-351X

David R. McKinzey, Shivasankari Gomathinayagam, Wezley C. Griffin, Kathleen N. Klinzing, Elizabeth P. Jeffries, Aleksandar Rajkovic, Michael A. Trakselis,

PARP inhibition in cancer therapy

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. 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. Homologous recombination (HR) of DNA involves multifaceted processes and pathways that respond to various types of DNA damage agents encountered during S/G2 phases of mitotic cells (1Wright W.D. Shah S.S. Heyer W.D. Homologous recombination and the repair of DNA double-strand breaks.J. Biol. Chem. 2018; 293: 10524-10535Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar, 2Quinet A. Lemacon D. Vindigni A. Replication fork reversal: Players and guardians.Mol. Cell. 2017; 68: 830-833Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Recombination occurring during meiosis can generate crossovers for genetic diversity and proper segregation in germline cells, utilizing many of the same enzymes (3Hunter N. Meiotic recombination: The essence of heredity.Cold Spring Harb. Perspect. Biol. 2015; 7a016618PubMed Google Scholar, 4Crickard J.B. Greene E.C. The biochemistry of early meiotic recombination intermediates.Cell Cycle. 2018; 17: 2520-2530Crossref PubMed Scopus (9) Google Scholar). Therefore, HR is vital for genomic integrity and diversity required for organismal survival. Defects in either mitotic or meiotic HR can directly contribute to increased cancer susceptibility and infertility through improper chromosomal rearrangements that represent incomplete intermediates and are hallmarks of disease. Various DNA helicases contribute to several steps in the recombination pathways either facilitating or dissolving hybrid DNA recombinants (5Huselid E. Bunting S.F. The regulation of homologous recombination by helicases.Genes (Basel). 2020; 11: 498Crossref Scopus (10) Google Scholar). Their individualized roles and substrate specificities in HR are commonly overlapping, making absolute distinctions of function difficult. MCM8 and MCM9 are recent additions to the roster of DNA helicases involved in HR (6Griffin W.C. Trakselis M.A. The MCM8/9 complex: A recent recruit to the roster of helicases involved in genome maintenance.DNA Repair (Amst.). 2019; 76: 1-10Crossref PubMed Scopus (21) Google Scholar). They are members of the ATPases associated with a variety of cellular activities (AAA+) superfamily and the minichromosome maintenance (MCM) family of proteins that includes MCM2-7 as the heterohexameric helicase complex central to the replication fork. The MCM8/9 complex does not appear to interact directly with MCM2-7, nor is it essential for replication (7Gambus A. Blow J.J. Mcm8 and Mcm9 form a dimeric complex in Xenopus laevis egg extract that is not essential for DNA replication initiation.Cell Cycle. 2013; 12: 1225-1232Crossref PubMed Scopus (17) Google Scholar). However, MCM8/9 is commonly associated with the replication fork (8Dungrawala H. Rose K.L. Bhat K.P. Mohni K.N. Glick G.G. Couch F.B. Cortez D. The replication checkpoint prevents two types of fork collapse without regulating replisome stability.Mol. Cell. 2015; 59: 998-1010Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) and may be able to take over helicase activities upon depletion of MCM2-7 (9Natsume T. Nishimura K. Minocherhomji S. Bhowmick R. Hickson I.D. Kanemaki M.T. Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis.Genes Dev. 2017; 31: 816-829Crossref PubMed Scopus (27) Google Scholar), suggesting a more active and dynamic role in elongation. Mounting evidence suggests that MCM8/9 is itself a heterohexameric complex involved in mediating unknown aspects of fork progression and/or downstream HR (10Lee K.Y. Im J.S. Shibata E. Park J. Handa N. Kowalczykowski S.C. Dutta A. MCM8-9 complex promotes resection of double-strand break ends by MRE11-RAD50-NBS1 complex.Nat. Commun. 2015; 6: 7744Crossref PubMed Scopus (55) Google Scholar, 11Nishimura K. Ishiai M. Horikawa K. Fukagawa T. Takata M. Takisawa H. Kanemaki M.T. Mcm8 and Mcm9 form a complex that functions in homologous recombination repair induced by DNA interstrand crosslinks.Mol. Cell. 2012; 47: 511-522Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 12Park J. Long D.T. Lee K.Y. Abbas T. Shibata E. Negishi M. Luo Y. Schimenti J.C. Gambus A. Walter J.C. Dutta A. The MCM8-MCM9 complex promotes RAD51 recruitment at DNA damage sites to facilitate homologous recombination.Mol. Cell. Biol. 2013; 33: 1632-1644Crossref PubMed Scopus (70) Google Scholar, 13Hustedt N. Saito Y. Zimmermann M. Alvarez-Quilon A. Setiaputra D. Adam S. McEwan A. Yuan J.Y. Olivieri M. Zhao Y. Kanemaki M.T. Jurisicova A. Durocher D. Control of homologous recombination by the HROB-MCM8-MCM9 pathway.Genes Dev. 2019; 33: 1397-1415Crossref PubMed Scopus (19) Google Scholar). Knockouts or knockdowns of MCM8 and/or MCM9 in mice and humans cause sex-specific tumorigenesis, defects in HR processing, and sensitivities to DNA damaging agents (11Nishimura K. Ishiai M. Horikawa K. Fukagawa T. Takata M. Takisawa H. Kanemaki M.T. Mcm8 and Mcm9 form a complex that functions in homologous recombination repair induced by DNA interstrand crosslinks.Mol. Cell. 2012; 47: 511-522Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 12Park J. Long D.T. Lee K.Y. Abbas T. Shibata E. Negishi M. Luo Y. Schimenti J.C. Gambus A. Walter J.C. Dutta A. The MCM8-MCM9 complex promotes RAD51 recruitment at DNA damage sites to facilitate homologous recombination.Mol. Cell. Biol. 2013; 33: 1632-1644Crossref PubMed Scopus (70) Google Scholar, 13Hustedt N. Saito Y. Zimmermann M. Alvarez-Quilon A. Setiaputra D. Adam S. McEwan A. Yuan J.Y. Olivieri M. Zhao Y. Kanemaki M.T. Jurisicova A. Durocher D. Control of homologous recombination by the HROB-MCM8-MCM9 pathway.Genes Dev. 2019; 33: 1397-1415Crossref PubMed Scopus (19) Google Scholar, 14Lutzmann M. Grey C. Traver S. Ganier O. Maya-Mendoza A. Ranisavljevic N. Bernex F. Nishiyama A. Montel N. Gavois E. Forichon L. de Massy B. Mechali M. MCM8- and MCM9-deficient mice reveal gametogenesis defects and genome instability due to impaired homologous recombination.Mol. Cell. 2012; 47: 523-534Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 15Hartford S.A. Luo Y. Southard T.L. Min I.M. Lis J.T. Schimenti J.C. Minichromosome maintenance helicase paralog MCM9 is dispensible for DNA replication but functions in germ-line stem cells and tumor suppression.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 17702-17707Crossref PubMed Scopus (60) Google Scholar). This results in diminished DNA damage signaling as exhibited by decreased phosphorylated CHK1 (pCHK1) and increased double-strand breaks (DSBs) as indicated by H2AX foci in the presence of various fork stalling or cross-linking agents. The absence of functional MCM8 or 9 impairs HR mediated fork rescue after damage through decreased recruitment of Mre11/Rad50/Nbs1 (MRN), RPA, and RAD51. In fact, MCM8/9 has been shown to be required for MRN nuclease activity to generate single-strand DNA (ssDNA) for HR after treatment with cisplatin (Cis-Pt) (10Lee K.Y. Im J.S. Shibata E. Park J. Handa N. Kowalczykowski S.C. Dutta A. MCM8-9 complex promotes resection of double-strand break ends by MRE11-RAD50-NBS1 complex.Nat. Commun. 2015; 6: 7744Crossref PubMed Scopus (55) Google Scholar). Even so, there are differing reports on the temporal association of MCM8/9 in relation to RAD51 after treatment with various DNA damage agents (9Natsume T. Nishimura K. Minocherhomji S. Bhowmick R. Hickson I.D. Kanemaki M.T. Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis.Genes Dev. 2017; 31: 816-829Crossref PubMed Scopus (27) Google Scholar, 10Lee K.Y. Im J.S. Shibata E. Park J. Handa N. Kowalczykowski S.C. Dutta A. MCM8-9 complex promotes resection of double-strand break ends by MRE11-RAD50-NBS1 complex.Nat. Commun. 2015; 6: 7744Crossref PubMed Scopus (55) Google Scholar, 11Nishimura K. Ishiai M. Horikawa K. Fukagawa T. Takata M. Takisawa H. Kanemaki M.T. Mcm8 and Mcm9 form a complex that functions in homologous recombination repair induced by DNA interstrand crosslinks.Mol. Cell. 2012; 47: 511-522Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 12Park J. Long D.T. Lee K.Y. Abbas T. Shibata E. Negishi M. Luo Y. Schimenti J.C. Gambus A. Walter J.C. Dutta A. The MCM8-MCM9 complex promotes RAD51 recruitment at DNA damage sites to facilitate homologous recombination.Mol. Cell. Biol. 2013; 33: 1632-1644Crossref PubMed Scopus (70) Google Scholar). These differences may be related to differential activities of RAD51 in HR-mediated fork stability/restart compared with that of direct DSB repair (16Bhat K.P. Cortez D. RPA and RAD51: Fork reversal, fork protection, and genome stability.Nat. Struct. Mol. Biol. 2018; 25: 446-453Crossref PubMed Scopus (126) Google Scholar) from specific DNA damage agents utilized or different eukaryotic cell types. Mutations in MCM8 and MCM9 in humans are linked to premature ovarian failure (POF) (17AlAsiri S. Basit S. Wood-Trageser M.A. Yatsenko S.A. Jeffries E.P. Surti U. Ketterer D.M. Afzal S. Ramzan K. Faiyaz-Ul Haque M. Jiang H. Trakselis M.A. Rajkovic A. Exome sequencing reveals MCM8 mutation underlies ovarian failure and chromosomal instability.J. Clin. Invest. 2015; 125: 258-262Crossref PubMed Scopus (131) Google Scholar, 18Wood-Trageser M.A. Gurbuz F. Yatsenko S.A. Jeffries E.P. Kotan L.D. Surti U. Ketterer D.M. Matic J. Chipkin J. Jiang H. Trakselis M.A. Topaloglu A.K. Rajkovic A. MCM9 mutations are associated with ovarian failure, short stature, and chromosomal instability.Am. J. Hum. Genet. 2014; 95: 754-762Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), amenorrhea, sterility (19Desai S. Wood-Trageser M. Matic J. Chipkin J. Jiang H. Bachelot A. Dulon J. Sala C. Barbieri C. Cocca M. Toniolo D. Touraine P. Witchel S. Rajkovic A. MCM8 and MCM9 nucleotide variants in women with primary ovarian insufficiency.J. Clin. Endocrinol. Metab. 2017; 102: 576-582PubMed Google Scholar), and cancer (20Goldberg Y. Halpern N. Hubert A. Adler S.N. Cohen S. Plesser-Duvdevani M. Pappo O. Shaag A. Meiner V. Mutated MCM9 is associated with predisposition to hereditary mixed polyposis and colorectal cancer in addition to primary ovarian failure.Cancer Genet. 2015; 208: 621-624Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In fact, deficiencies in MCM8 or 9 are phenotypically similar to Fanconi anemia (FA) patient mutations (21Giampietro P.F. Adler-Brecher B. Verlander P.C. Pavlakis S.G. Davis J.G. Auerbach A.D. The need for more accurate and timely diagnosis in Fanconi anemia: A report from the International Fanconi Anemia Registry.Pediatrics. 1993; 91: 1116-1120PubMed Google Scholar) (where ∼50% of patients are infertile (22Alter B.P. Frissora C.L. Halperin D.S. Freedman M.H. Chitkara U. Alvarez E. Lynch L. Adler-Brecher B. Auerbach A.D. Fanconi's anaemia and pregnancy.Br. J. Haematol. 1991; 77: 410-418Crossref PubMed Scopus (65) Google Scholar)) and suggest an overlapping role in interstrand cross-link (ICL)-coupled HR, but without the associated anemia. MCM9 mutations are also linked to hereditary mixed polyposis and colorectal cancer, commonly caused by loss of function in DNA mismatch repair (MMR) (23Poulogiannis G. Frayling I.M. Arends M.J. DNA mismatch repair deficiency in sporadic colorectal cancer and Lynch syndrome.Histopathology. 2010; 56: 167-179Crossref PubMed Scopus (154) Google Scholar, 24Traver S. Coulombe P. Peiffer I. Hutchins J.R. Kitzmann M. Latreille D. Mechali M. MCM9 is required for mammalian DNA mismatch repair.Mol. Cell. 2015; 59: 831-839Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The MMR connection may be more important in regulating microhomology mediated HR that requires mismatch repair for Holliday junction progression (24Traver S. Coulombe P. Peiffer I. Hutchins J.R. Kitzmann M. Latreille D. Mechali M. MCM9 is required for mammalian DNA mismatch repair.Mol. Cell. 2015; 59: 831-839Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 25Tham K.C. Kanaar R. Lebbink J.H.G. Mismatch repair and homeologous recombination.DNA Repair (Amst.). 2016; 38: 75-83Crossref PubMed Scopus (53) Google Scholar, 26Spies M. Fishel R. Mismatch repair during homologous and homeologous recombination.Cold Spring Harb. Perspect. Biol. 2015; 7: a022657Crossref PubMed Scopus (78) Google Scholar) or from overlapping recognition of duplex distorting lesions (27Kato N. Kawasoe Y. Williams H. Coates E. Roy U. Shi Y. Beese L.S. Scharer O.D. Yan H. Gottesman M.E. Takahashi T.S. Gautier J. Sensing and processing of DNA interstrand crosslinks by the mismatch repair pathway.Cell Rep. 2017; 21: 1375-1385Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 28Yang W. Structure and mechanism for DNA lesion recognition.Cell Res. 2008; 18: 184-197Crossref PubMed Scopus (99) Google Scholar). Instead, several human cancer genomes show homo and heterozygous deletions in MCM9 coding regions, many missense mutations in MCM8 and MCM9, and altered expression levels that correlate with aggressive clinical features and poorer long-term survival in several human cancers (10Lee K.Y. Im J.S. Shibata E. Park J. Handa N. Kowalczykowski S.C. Dutta A. MCM8-9 complex promotes resection of double-strand break ends by MRE11-RAD50-NBS1 complex.Nat. Commun. 2015; 6: 7744Crossref PubMed Scopus (55) Google Scholar, 29Kim J.H. Dhanasekaran S.M. Mehra R. Tomlins S.A. Gu W. Yu J. Kumar-Sinha C. Cao X. Dash A. Wang L. Ghosh D. Shedden K. Montie J.E. Rubin M.A. Pienta K.J. et al.Integrative analysis of genomic aberrations associated with prostate cancer progression.Cancer Res. 2007; 67: 8229-8239Crossref PubMed Scopus (104) Google Scholar, 30Sung C.O. Kim S.C. Karnan S. Karube K. Shin H.J. Nam D.H. Suh Y.L. Kim S.H. Kim J.Y. Kim S.J. Kim W.S. Seto M. Ko Y.H. Genomic profiling combined with gene expression profiling in primary central nervous system lymphoma.Blood. 2011; 117: 1291-1300Crossref PubMed Scopus (71) Google Scholar, 31He D.M. Ren B.G. Liu S. Tan L.Z. Cieply K. Tseng G. Yu Y.P. Luo J.H. Oncogenic activity of amplified miniature chromosome maintenance 8 in human malignancies.Oncogene. 2017; 36: 3629-3639Crossref PubMed Scopus (16) Google Scholar). Like that found for 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 HR repair that can be exploited in the clinic for synthetically lethal therapies with platinum-based DNA cross-linking agents (32Morii I. Iwabuchi Y. Mori S. Suekuni M. Natsume T. Yoshida K. Sugimoto N. Kanemaki M.T. Fujita M. Inhibiting the MCM8-9 complex selectively sensitizes cancer cells to cisplatin and olaparib.Cancer Sci. 2019; 110: 1044-1053Crossref PubMed Scopus (11) Google Scholar). MCM9 contains a unique and large C-terminal extension (CTE) not found in the other MCM family members and can be alternatively spliced to give a shorter MCM9M isoform that retains the conserved helicase domains but removes the CTE (33Jeffries E.P. Denq W.I. Bartko J.C. Trakselis M.A. Identification, quantification, and evolutionary analysis of a novel isoform of MCM9.Gene. 2013; 519: 41-49Crossref PubMed Scopus (6) Google Scholar). The CTE is a common feature in other HR helicases and is generally considered to be unstructured with scattered putative amino acid motifs that can impact protein interactions and affect proper function (6Griffin W.C. Trakselis M.A. The MCM8/9 complex: A recent recruit to the roster of helicases involved in genome maintenance.DNA Repair (Amst.). 2019; 76: 1-10Crossref PubMed Scopus (21) Google Scholar). However, no such motifs or role for the CTE has been identified for MCM9. Here, we can show that the CTE in MCM9 plays an essential role in nuclear import and formation of DNA repair foci after treatment with the cross-linking agent, mitomycin C (MMC). We have identified and validated a unique "bipartite-like" nuclear localization signal (NLS) within the CTE that directs the nuclear import of MCM8. Finally, we have also identified a BRCv motif that is required for the recruitment of RAD51 to sites of MMC-induced damage that is analogous to that found in other HR helicases (34Islam M.N. Paquet N. Fox 3rd, D. Dray E. Zheng X.F. Klein H. Sung P. Wang W. A variant of the breast cancer type 2 susceptibility protein (BRC) repeat is essential for the RECQL5 helicase to interact with RAD51 recombinase for genome stabilization.J. Biol. Chem. 2012; 287: 23808-23818Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The overall results confirm an influential role for the CTE of MCM9 in importing the MCM8/9 complex into the nucleus, directing it to sites of cross-link damage, and recruiting RAD51 for downstream repair. We and others have shown previously that MCM8 and MCM9 are generally localized to the nucleus and form nuclear foci after damage with MMC (11Nishimura K. Ishiai M. Horikawa K. Fukagawa T. Takata M. Takisawa H. Kanemaki M.T. Mcm8 and Mcm9 form a complex that functions in homologous recombination repair induced by DNA interstrand crosslinks.Mol. Cell. 2012; 47: 511-522Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 17AlAsiri S. Basit S. Wood-Trageser M.A. Yatsenko S.A. Jeffries E.P. Surti U. Ketterer D.M. Afzal S. Ramzan K. Faiyaz-Ul Haque M. Jiang H. Trakselis M.A. Rajkovic A. Exome sequencing reveals MCM8 mutation underlies ovarian failure and chromosomal instability.J. Clin. Invest. 2015; 125: 258-262Crossref PubMed Scopus (131) Google Scholar). However, we sought to examine which of the domains of MCM9 (Fig. 1A) are required for nuclear foci formation after MMC treatment. MCM9 contains a unique CTE that comprises 42% of the 1143 amino acids and 52 kDa of the total 127 kDa molecular weight of the full-length protein. The CTE is more hydrophilic compared with the rest of the protein, has a higher disorder probability, and is likely mostly unstructured (Fig. S1). As the CTE of MCM9 is predicted to have high disorder and low secondary structure, we sought to directly measure the solution structure composition of various MCM9 CTE truncations (Fig. 1B) using circular dichroism (CD). Spectra for both MCM9 643 to 900 and 680 to 900 show a pronounced minimum at 201 nm (Fig. 1C), which is consistent with significant disorder (35Chemes L.B. Alonso L.G. Noval M.G. de Prat-Gay G. Circular dichroism techniques for the analysis of intrinsically disordered proteins and domains.Methods Mol. Biol. 2012; 895: 387-404Crossref PubMed Scopus (56) Google Scholar). There are shallow valleys from 215 to 230 nm indicative of minor α-helical and antiparallel β-sheet characteristics, but the overall spectrum is representative of a highly disordered (∼50%) protein, indicated as "other" in the quantification (Fig. 1D). For the limited secondary structure, the two truncations are highly similar with only ∼7% helical and ∼30% β-sheet. Full-length MCM9-GFP (MCM9L) is nuclear and forms a significant number of nuclear foci upon treatment with the cross-linking agent MMC (Fig. 2, A–C). Interestingly, the transfection of the alternatively spliced MCM9 product (MCM9M) (33Jeffries E.P. Denq W.I. Bartko J.C. Trakselis M.A. Identification, quantification, and evolutionary analysis of a novel isoform of MCM9.Gene. 2013; 519: 41-49Crossref PubMed Scopus (6) Google Scholar) showed primarily cytoplasmic staining, while MCM9Cterm showed concise nuclear staining but an absence of repair foci with MMC. Nuclear and cytoplasmic extractions of various MCM9-GFP transfected constructs were used to validate these observations in a population of cells and quantified by calculating nuclear/cytoplasmic (N/C) ratios (Fig. 2D). MCM9L is more nuclear than cytoplasmic; MCM9M is primarily cytoplasmic; and MCM9Cterm is more nuclear, while alpha-Tubulin and Lamin B1 were used as cytoplasmic and nuclear controls, respectively. Quantification of the nuclear/cytoplasmic (N/C) ratios show values above 1 for both MCM9L and MCM9Cterm but below 1 for MCM9M that lacks the CTE, which are statistically different. Although MCM9Cterm does not form MMC-induced foci, it does interact with chromatin probably though the winged-helix domain contained in the CTE (Fig. S2). Therefore, the CTE directs MCM9 into the nucleus, but the helicase core is required for localization to DNA repair sites. As MCM9Cterm had predominantly nuclear staining but MCM9M did not, we searched for nuclear localization sequences (NLS) in the CTE and found four conserved, high-confidence putative NLS sequences (pNLS) (Fig. S3). These four pNLS sequences were individually mutated in MCM9L-GFP, transfected into U2OS cells, and the localization was examined by confocal microscopy (Fig. 3, A and B). U2OS cells were used because of their larger nuclei that can be easily distinguished from cytoplasmic areas. Mutation of pNLS1 or pNLS2 resulted in primarily cytoplasmic staining indicating a lack of nuclear import. Alternatively, mutation of pNLS3 and pNLS4 had no noticeable effect on nuclear import. Interestingly, mutation of either pNLS1 or pNLS2 alone had a clear effect on nuclear import, indicating that a bipartite NLS1/NLS2 is required; however, the linker between NLS1 and NLS2 spans 67 a.a., much longer than a canonical bipartite NLS that generally has a 10 to 12 a.a. linker. Therefore, the import signal motif for MCM9 is "bipartite-like" consisting of dual NLS1/NLS2 motifs connected by an extended linker region. To unequivocally show the effect of mutating NLS1 or NLS2 in a population of cells, we performed nuclear and cytosolic extractions on MCM9L-GFP construct transfected into 293T cells (Fig. 3C). Again, wild-type (WT) MCM9L is confined more in the nuclear fraction than the cytoplasm as indicated by N/C = 1.5. However, 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. We also searched in silico for a pNLS in MCM8, but no high-confidence motifs were detected. Therefore, we hypothesized that the NLS1/2 in the CTE of MCM9 may be responsible for nuclear import of the MCM8/9 complex. Therefore, we created custom CRISPR-Cas9 MCM8 or MCM9 knockout cell lines from 293T cells with significant multiallelic indels and validated phenotypically with severe MMC sensitivity (Fig. S4). To confirm if knockout of MCM9 disrupts import of MCM8 into the nucleus, MCM8-GFP was transfected into the MCM9KO cell line (Fig. 4B) . In WT 293T cells, MCM8-GFP is primarily nuclear and forms foci with MMC treatment (Fig.4A); however, in MCM9KO cells, the MCM8-GFP staining is diffuse throughout the cytoplasm, not localized to the nucleus, and fails to form MMC-induced nuclear foci (Fig. 4B). Transfection of MCM9L-GFP or MCM9M-GPF into MCM8KO cells (Fig. 4C) shows essentially the same results (as in Fig. 2, B and C) with nuclear and cytoplasmic staining, respectively. Therefore, MCM8 is imported into the nucleus complexed with MCM9 as directed by the "bipartite-like" NLS1/NLS2. The BRC repeat sequence is a structural motif in the breast cancer type 2 susceptibility protein (BRCA2) that is utilized to promote RAD51 nucleoprotein filament formation during fork reversal or recombination (36Shivji M.K. Mukund S.R. Rajendra E. Chen S. Short J.M. Savill J. Klenerman D. Venkitaraman A.R. The BRC repeats of human BRCA2 differentially regulate RAD51 binding on single- versus double-stranded DNA to stimulate strand exchange.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 13254-13259Crossref PubMed Scopus (54) Google Scholar). The eight repeats in BRCA2 show a consensus BRC motif sequence (Fig. S5), and variants of these BRC motifs (BRCv) have been found in other proteins including RECQL5 and yeast helicases Srs2, Sgs1, Mph1, and Pif1 (34Islam M.N. Paquet N. Fox 3rd, D. Dray E. Zheng X.F. Klein H. Sung P. Wang W. A variant of the breast cancer type 2 susceptibility protein (BRC) repeat is essential for the RECQL5 helicase to interact with RAD51 recombinase for genome stabilization.J. Biol. Chem. 2012; 287: 23808-23818Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The BRCv motif in RECQL5 is shown to be important for interacting with RAD51, directing D-loop and filament formation, and responding to cross-linking stress. In our examination of the expansive and unique CTE of MCM9, we also identified a putative BRCv motif that is similar to that found in RECQL5 and adjacent to a predicted WH motif important for DNA binding (Fig. 5, A and B). The BRCv consensus sequence within motif 1, FxTASxxxϕxϕS, is highly similar to the BRC repeats themselves FxTASGKxϕxϕS (where "ϕ" represents hydrophobic residues). BRCv motif 2 occurs after a linker region in these HR helicases that is not present in BRCA2 and is less well conserved. However, a small hydrophobic/electrostatic patch in Motif 2, ϕL(D/E)-(D/E), is present in MCM9 and similar to RECQL5. Both BRCv motifs 1 and 2 in MCM9 are well conserved across Mammalia species, but less conserved in other metazoans (Fig. S5B). Structural modeling of the MCM9 BRCv motif threaded onto BRCA2-BRC4 component in complex with RAD51 (34Islam M.N. Paquet N. Fox 3rd, D. Dray E. Zheng X.F. Klein H. Sung P. Wang W. A variant of the breast cancer type 2 susceptibility protein (BRC) repeat is essential for the RECQL5 helicase to interact with RAD51 recombinase for genome stabilization.J. Biol. Chem. 2012; 287: 23808-23818Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) shows the importance of the universally conserved F687 residue binding deep into a hydrophobic pocket of RAD51 (Fig. 5C and Fig. S5C). Other conserved residues (T689, S690, S691, I695, and Y697) within motif 1 make backbone and side-chain contacts across a path of hydrophobic surface area. Residues within motif 2 make both hydrophobic and hydrogen bonding contacts with RAD51 that would loop out the linker region in between (Fig. S5, D and E). Leu714 is predicted to make hydrophobic contacts with RAD51 residues 254-5 and 258-9. Our model also predicts that Glu722 and Asn724 make H-bond contacts with Tyr205 and Arg250 of RAD51, respectively. Together MCM9 BRCv motifs 1 and 2 are predicted to make sufficient hydrophobic surface area contacts with RAD51 that are anchored by F687. To test whether mutation of the MCM9 BRCv motif has any effect on foci formation in MMC treated cells, we transfected mutated MCM9-GFP constructs (FR687/8AA, F687E, and L720A) into 293T cells (Fig. 5, D and E). The GFP fluorescence for the BRCv mutants was nuclear as expected, but MMC did not induce significant foci formation in any of these mutants. Quantification of cells with >5 foci with MMC treatment for WT (78.7 ± 8.0%) versus BRCv− mutants: F687A/R688A (12.2 ± 0.8%), F687E (20.2 ± 0.8%), and L687A (10.4 ± 0.5%) show significant differences (Fig. 5E) and illustrate the importance of the bipartite BRCv motif in directing downstream DNA repair. As both BRCA1 and RAD51 form MMC-induced nuclear foci (37Pichierri P. Averbeck D. Rosselli F. DNA cross-link-dependent RAD50/MRE11/NBS1 subnuclear assembly requires the Fanconi anemia C protein.Hum. Mol.