P326 DNA replication initiator proteins stabilize the kinetochore in the human fungal pathogen Cryptococcus neoformans

POSTER SESSION 3, SEPTEMBER 23, 2022, 12:30 PM - 1:30 PM: OBJECTIVES: DNA replication licensing ensures maintenance of the ploidy state by limiting DNA replication once per cell cycle. The mini-chromosome maintenance (MCM) complex is an evolutionarily conserved DNA helicase in eukaryotes that aids this process. Upon activation in the S phase by several protein kinases, the MCM complex unwinds the double-stranded DNA and moves away from DNA replication origins preventing re-initiation of replication in the remainder of the cell cycle. Deregulation of MCM function can lead to malignant transformation of proliferating cells as indicated by their upregulated expression in human cancer and pre-cancerous cells. MCMs, therefore, serve as important biomarkers and potential targets for anti-cancer drugs. This study aims to decipher the effect of deregulated expression of the MCM complex on the cell cycle events of the human fungal pathogen, Cryptococcus neoformans, which is more similar to metazoans than other budding yeast. Aneuploidy-mediated drug resistance is a common mechanism in many major human fungal pathogens including Cryptococcus and Candida. METHODS: We generated conditional mutants of individual subunits of the MCM complex in C. neoformans and assayed the impact of their altered expression on cell cycle events. We also tested the alternative role of MCM subunits in the assembly of the kinetochore, a vital component of the chromosome segregation machinery. RESULTS: Our screen with MCM conditional mutants identified two in vivo functional subcomplexes that comprise the MCM complex in C. neoformans. Although upregulated expression of either Mcm2 or Mcm3 does not affect cell cycle progression, overexpression of either Mcm6 or Mcm7 led to the accumulation of cells in the large bud stage with nuclear segregation defects. This work provides evidence for the first time for a mitotic role of pre-replication complex proteins, MCM 2-7 complex in Cryptococcus. Depletion of Mcm2 led to arrest of cells in the S and G2/M stages of the cell cycle with defects in nuclear segregation. Localization and expression of several kinetochore proteins upon depletion of Mcm2 established that Mcm2 is vital for kinetochore assembly/integrity. Although the centromeric histone H3 variant, CENP-A, remains largely clustered, the outer kinetochore did not mature indicating that Mcm2 alone or as part of the MCM complex plays a role in kinetochore assembly/integrity and thereby in chromosome segregation. CONCLUSION: A conserved eukaryotic DNA helicase, MCM 2-7 complex, has an unexplored non-canonical role in kinetochore assembly/integrity and chromosome segregation.