Molecular and func-onal analysis of Cdc14 and PP4 phosphatases in response to DNA damage.

Abstract

[ENG]Cells are constantly exposed to different endogenous and exogenous agents that can produce lesions in the gene7c material. Within all the types of DNA damage, double-strand breaks are the most damaging type of injury to the cell. In response to this damage, cells have developed a set of processes collec7vely known as the DNA Damage Response (DDR). One of the ini7al steps in homologous recombina7on (HR) is DNA-end resec7on. It involves the degrada7on of one DNA strand on both sides of the DSB in the 5'-3’ sense, genera7ng single-stranded DNA (ssDNA). Subsequently, different proteins bind to this ssDNA forming a protofilament capable of searching for homology and invading the template strand. While many proteins involved in these processes are regulated through phosphoryla7on by protein kinases, litle is known about the role of protein phosphatases in these mechanisms. The role of cyclin-dependent kinase (CDK) in regula7ng numerous mechanisms in response to DNA damage has been extensively studied. Since Cdc14 is the only known phosphatase that antagonizes Cdk, it is reasonable to think that its func7on is also crucial in regula7ng these processes. Thus, the absence of Cdc14 ac7vity lead s to excessive resec7on and genomic instability, sugges7ng a potencial role for the phosphatase in nega7vely regula7ng this process to temporally restrain resec7on progression and ensure an accurate DNA repair via homologous recombina7on. Addi7onally, high-throughput sequencing analyses have allowed for the inves7ga7on of various aspects of DNA repair at the nucleo7de level, including the extent of resec7on, its direc7onality, and the gene conversion events generated during lesion repair. Interes7ngly, the removal of Dna2 ac7vity or the disrup7on of its Cdk phosphoryla7on sites prevents the over-resec7on phenotype observed in the absence of the phosphatase, sugges7ng that Cdc14 nega7vely regulates resec7on by controlling the Sgs1/Dna2 complex. Curiously, only the mito7cally ac7vated Cdc14 (FEAR and MEN), but not DNA damage-dependent Cdc14 ac7va7on, dephopshorylates Dna2. Addi7onally, the dissolu7on of Dna2 foci, as observed by fluorescence microscopy, occurs exclusively during mitosis, sugges7ng that only mito7c ac7va7on of Cdc14 inhibits resec7on. In summary, these results demonstrate that the Cdk/Cdc14 module func7ons as a molecular switch that acts on the exonuclease Dna2 to regulate the proper extension of singlestranded DNA ends generated during resec7on, ensuring the accurate restora7on of the DNA molecule.