Abstract
DNA replication, recombination, and repair are highly interconnected processes the disruption of which must be coordinated in cancer. HERC2, a large HECT protein required for homologous recombination repair, is an E3 ubiquitin ligase that targets breast cancer suppressor BRCA1 for degradation. Here, we show that HERC2 is a component of the DNA replication fork complex that plays a critical role in DNA elongation and origin firing. In the presence of BRCA1, endogenous HERC2 interacts with Claspin, a protein essential for G(2)-M checkpoint activation and replication fork stability. Claspin depletion slowed S-phase progression and additional HERC2 depletion reduced the effect of Claspin depletion. In addition, HERC2 interacts with replication fork complex proteins. Depletion of HERC2 alleviated the slow replication fork progression in Claspin-deficient cells, suppressed enhanced origin firing, and led to a decrease in MCM2 phosphorylation. In a HERC2-dependent manner, treatment of cells with replication inhibitor aphidicolin enhanced MCM2 phosphorylation. Taken together, our results suggest that HERC2 regulates DNA replication progression and origin firing by facilitating MCM2 phosphorylation. These findings establish HERC2 as a critical function in DNA repair, checkpoint activation, and DNA replication.
Highlights
DNA replication, recombination, and repair coordinately maintain genome stability, and their defect is a hallmark of cancer cells
Because depletion of HERC2 compensated for BRCA1 instability in BARD1-deficient cells and restored G2–M checkpoint function, we propose that HERC2 inhibits G2–M checkpoint function by destabilizing BRCA1
HERC2 interacts with Claspin and affects S-phase progression
Summary
DNA replication, recombination, and repair coordinately maintain genome stability, and their defect is a hallmark of cancer cells. We show that HERC2 is a component of replication fork complex and regulates the fork progression and origin firing in conjunction with Claspin. DNA fiber experiments Forty-eight hours after siRNA transfection, cells were pulse labeled with 25 mmol/L BrdUrd for indicated time length, followed by 250 mmol/L iododeoxyuridine (IdUrd) for 20 minutes.
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