Exploring the functions of the CST complex in the DNA damage response

DNA replication stress is an important source of genomic instability, characterized by the transient slowing or stalling of replication forks due to damaged DNA, unusual DNA structures, repetitive sequences, or nucleotide depletion. Stalled forks typically lead to the uncoupling between leading and lagging-strand polymerases or polymerases from helicases. This block generates single-stranded DNA regions covered by replication protein A (RPA), which recruits, in the yeast S. cerevisiae, the checkpoint kinase Mec1. Once activated, Mec1 triggers the downstream kinase Rad53, which regulates cell cycle progression, increases dNTP pools, and prevents fork collapse. The S-phase checkpoint is crucial for cell survival during replication stress, aiding in the resumption of DNA synthesis once fork blockage is relieved. Nucleases such as Mre11, Exo1, and Dna2-Sgs1 process replication intermediates to recover stalled replication forks. This process must be tightly regulated, as an unscheduled nuclease activity could destroy the replication fork structure, leading to genome instability. To better understand how these pathways are regulated, we performed a genetic screen aimed to identify mutations that suppress the DNA damage sensitivity of mec1 mutant cells. Through genome-wide sequencing and genetic analyses, we identified a suppressor mutation in the STN1 gene. Stn1 is part of the RPA-like Cdc13-Stn1-Ten1 (CST) complex, which recognizes single-stranded DNA and is known to be involved in telomere maintenance. We also found that the absence of the Stn1 C-terminus exacerbates the sensitivity of mec1 mutant cells to DNA damage. These results suggest that Stn1 supports the functions of the checkpoint kinase Mec1 under DNA replication stress. We will present data regarding the underlying molecular mechanism.