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 minichromosome maintenance (MCM) 8/9 helicase is a AAA+ complex involved in DNA replication-associated repair. Despite high sequence homology to the MCM2-7 helicase, a precise cellular role for MCM8/9 has remained elusive. We have interrogated the DNA synthesis ability and replication fork stability in cells lacking MCM8 or 9 and find that there is a functional partitioning of MCM8/9 activity between promoting replication fork progression and protecting persistently stalled forks. The helicase function of MCM8/9 aids in normal replication fork progression, but upon persistent stalling, MCM8/9 directs additional downstream stabilizers, including BRCA1 and Rad51, to protect forks from excessive degradation. Loss of MCM8 or 9 slows the overall replication rate and allows for excessive nascent strand degradation, detectable by increased markers of genomic damage. This evidence defines multifunctional roles for MCM8/9 in promoting normal replication fork progression and genome integrity following stress.
Efficiency of DNA replication is crucial to cell survival. Recognition of DNA damage by replication complexes results in impedance of this process until the damage is repaired. Fork stalling and reversal are important for the repair of DNA lesions. Stalled forks must be resolved quickly in order to prevent fork collapse resulting in double‐strand breaks (DSBs). Several proteins are recruited to mitigate damage to DNA prior to DSB formation. MCM8/9 is believed to play a role in both protection of DNA at stalled forks and in repair of DSBs through a helicase activity. Mutation of MCM8 or MCM9 has been attributed to development of cancer and infertility. The MCM8/9 complex recruits Rad51 to double stranded break sites, but its role in fork stalling remains unknown. A CRISPR‐Cas9 generated MCM9 knockout cell line was used to assess MCM8/9‐mediated protection of stalled or reversed forks from nucleolytic degradation using DNA fiber assays. Two halogenated thymine analogs, CldU and IdU, are added to the nucleotide pool to monitor discrete DNA replication tracks within HEK293T cells. After CldU and IdU pulses, DNA replication is stalled through the addition of hydroxyurea (HU) to allow for fork reversal. The IdU/CldU length ratio measured from DNA fiber analysis can reveal fork protection status. MCM9 knockout cells had a significant reduction in IdU/CldU track ratios demonstrating its importance in maintaining replication fork stability following stalling by HU. These results suggest that MCM8/9 plays a crucial but unidentified role in maintenance of replication fork integrity and protection of stalled forks during replication. Current and future experiments will focus on the direct interactions and effects of knockdowns of other known fork protection proteins with MCM9 to more definitely determine MCM8/9 function in maintaining the human genome. Support or Funding Information This work is supported by the National Institutes of Health (NIH) GM135791.
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.
CD8+ T cells play crucial roles in cellular immune mechanisms that clear invading viruses and prevent reinfection. Virus specific CD8+ T cells expand approximately 10,000 fold, undergoing up to 20 cell divisions in the first week of infection. While this rapid expansion is needed to reduce viral loads, replication can lead to DNA damage and telomere shortening, resulting in growth arrest. T cells must be equipped with mechanisms that promptly promote DNA repair to protect cells from premature senescence and death. We recently determined that the E3 Ubiquitin ligase Cullin-4b (Cul4b) regulates genome integrity in CD4+ T cells by helping to resolve DNA damage. In keeping with this, aberrant expression of Cul4b and its ortholog Cul4a has been reported in many cancers. However, whether Cul4b is important in controlling CD8+ T cell numbers or viral clearance is not known. To address this, we infected control and Cul4bcKO mice with LCMV. Following infection, virus-specific CD8+ T cells lacking Cul4b failed to expand or acquire effector functions. As a consequence, Cul4bcKO mice were unable to clear LCMV. Furthermore, Cul4b-deficient CD8+ T cells were unable to differentiate into memory T cells. Using IP and tandem mass spectrometry, we found Cul4b associated with the chromatin remodeling BAF complex (SWI/SNF). Specifically, our data supports that Brg1, a component of SWI/SNF complex, is a client of the Cul4b ubiquitin ligase. Loss of Cul4b increased Brg1 binding to chromatin following CD8+ T cell activation, impacting gene expression and/or cell cycle arrest. Together, these data support that Cul4b limits the amount of Brg1 associated with chromatin, and allows the expansion and differentiation of CD8+ T cells thus promoting antiviral immunity.
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