MOLECULAR BASES OF THE GENOMIC INSTABILITY TRIGGERED BY INCOMPLETE MIDAS IN CHK1 DEFICIENT BACKGROUNDS

MARINA ALEJANDRA GONZÁLEZ BESTEIRO; VANESA GOTTIFREDI; NICOLAS CALZETTA

Workshop; 5th AEK Autum School Replication stress in cancer; 2020

German Cancer Society Experimental Cancer Research

Purpose: The S phase is the period of the cell cycle in which genomes are entirely duplicated. Butin organisms with gigabase-sized genomes, such as humans, cells routinely enter the G2 phasewith stretches of under-replicated DNA (UR-DNA). Such DNA regions can be duplicated in mitosisby a specialized DNA replication pathway known as mitotic DNA synthesis (MiDAS). Failure toreplicate UR-DNA during mitosis results in aberrant chromosome segregation, whichsubsequently leads to genomic instability. Genomic instability is defined as a persistent and highrate of mutations and is a hallmark of cancer; it contributes to intratumoral geneticheterogeneity, which implies the risk of developing resistance to oncologic treatments. Thus,elucidating the mechanisms that trigger genomic instability is of utmost importance to cancerresearch.Methods: To analyze chromosome mis-segregation in Chk1-deficient cells we measured thepercentage of cells with anaphase aberrations and micronuclei. MiDAS was analyzed by 45 minpulses of EdU, and DAPI staining served to visualize metaphases. We determined presence ofUR-DNA by the frequency of ultra-fine bridges (PICH staining) in anaphase and 53BP1-nuclearbodies in G1.Results: Herein, we show that downregulation of Checkpoint Kinase 1 (Chk1), a key mediator ofthe S-phase checkpoint and whose inhibitors are undergoing clinical evaluation across a varietyof cancers, induces UR-DNA and MiDAS. But in apparent contrast with the idea that MiDAScompletes DNA duplication and hence safeguards genomic stability, our data show that MiDAS inChk1-deficient cells induces chromosome mis-segregation. Importantly, we unveil the molecularbasis of aberrant MiDAS. Upon Chk1 loss, mitotic DNA replication intermediates in mitosis stalldue to nucleotide shortage. Stalled DNA replication intermediates are then cleaved by thestructure specific endonuclease Mus81-Eme1, and these mitotic DSBs culminate in chromosomemis-segregation. Intriguingly, both MiDAS abrogation, Mus81-Eme1 down-regulation and MiDASupregulation by nucleosides reverts the genomic instability caused by Chk1 depletion.Conclusion: Such observation indicates that genomic instability is the consequence ofincomplete duplication of UR-DNA by MiDAS. Our work unveils a novel molecular pathway leadingto genomic instability in cancer cells. Given the interest in avoiding genomic instability during