Role of MCM8/9 in DNA Fork Protection

Abstract

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.