Chk1 loss creates replication barriers that compromise cell survival independently of excess origin firing

Abstract

The effectiveness of checkpoint kinase 1 (Chk1) inhibitors at killing cancer cells is considered to be fully dependent on their effect on DNA replication initiation. Chk1 inhibition boosts origin firing, presumably limiting the availability of nucleotides and in turn provoking the slowdown and subsequent collapse of forks, thus decreasing cell viability. Here we show that slow fork progression in Chk1‐inhibited cells is not an indirect effect of excess new origin firing. Instead, fork slowdown results from the accumulation of replication barriers, whose bypass is impeded by CDK‐dependent phosphorylation of the specialized DNA polymerase eta (Polη). Also in contrast to the linear model, the accumulation of DNA damage in Chk1‐deficient cells depends on origin density but is largely independent of fork speed. Notwithstanding this, origin dysregulation contributes only mildly to the poor proliferation rates of Chk1‐depleted cells. Moreover, elimination of replication barriers by downregulation of helicase components, but not their bypass by Polη, improves cell survival. Our results thus shed light on the molecular basis of the sensitivity of tumors to Chk1 inhibition.

Mus81-Eme1–dependent aberrant processing of DNA replication intermediates in mitosis impairs genome integrity

“…We have recently identified excess chromatin binding of the helicase cofactor CDC45 as the cause of increased origin firing and asymmetric fork slowdown in Chk1-deficient cells ( 9 ). Partial depletion of CDC45 does not interfere with cell proliferation ( 9 , 30 , 31 ), but it does nullify the massive accumulation of DNA damage and DSBs in Chk1-depleted U2OS cells ( Fig. 1, A to C ).…”

“…In apparent contrast, our data show that MiDAS jeopardizes chromosome stability in a Chk1-deficient background. In cells deficient in the DDR components Chk1 and WEE1, the availability of nucleotides is restricted ( 8 , 9 , 38 , 41 , 42 ). In WEE1-inhibited, but not in Chk1-inhibited, cells, nucleotide scarcity triggers replication catastrophe and chromosome pulverization ( 8 , 9 , 37 , 38 ).…”

“…Our data also show a dissection between chromosome mis-segregation and replication catastrophe. Replication catastrophe, which is characterized by pan-nuclear accumulation of γH2AX and single-stranded DNA, leads to cell death in S phase ( 7 – 9 , 11 , 30 , 38 , 41 ). So, we reasoned that chromosome mis-segregation might not precede cell death in Chk1-deficient cells.…”

“…The ATR-Chk1 pathway adjusts the nucleotide pool via control of RRM2, a regulatory subunit of the ribonucleotide reductase, which catalyzes the rate-limiting step for deoxynucleoside triphosphate (dNTP) production ( 41 ). Chk1-inhibited cells show reduced dNTP levels ( 42 ), resulting in the slowdown of replication forks ( 8 , 9 ). Notwithstanding this, we and others have been unable to detect an effect of such nucleotide deficiency on the DNA damage load in S phase ( 8 , 9 , 37 ).…”

Inducible SMARCAL1 knockdown in iPSC reveals a link between replication stress and altered expression of master differentiation genes

“…4A). To more accurately evaluate the presence of fork obstacles accumulating with time, we also used a longer IdU pulse (González Besteiro et al, 2019). Analysis of IdU track length in dual-labelled fibres showed that loss of SMARCAL1 did not reduce fork speed in iPSCs at 15 min, although it significantly decreased the length of IdU tracks at 30 min (Fig.…”