Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint.

By T. Byun, Marcin Pacek, M. Yee, Johannes C. Walter, Karlene A. Cimprich. Genes & Development, 2005

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The ATR-dependent DNA damage response pathway can respond to a diverse group of lesions as well as inhibitors of DNA replication. Using the Xenopus egg extract system, we show that lesions induced by UV irradiation and cis-platinum cause the functional uncoupling of MCM helicase and DNA polymerase activities, an event previously shown for aphidicolin. Inhibition of uncoupling during elongation with inhibitors of MCM7 or Cdc45, a putative helicase cofactor, results in abrogation of Chk1 phosphorylation, indicating that uncoupling is necessary for activation of the checkpoint. However, uncoupling is not sufficient for checkpoint activation, and DNA synthesis by Polalpha is also required. Finally, using plasmids of varying size, we demonstrate that all of the unwound DNA generated at a stalled replication fork can contribute to the level of Chk1 phosphorylation, suggesting that uncoupling amplifies checkpoint signaling at each individual replication fork. Taken together, these observations indicate that functional uncoupling of MCM helicase and DNA polymerase activities occurs in response to multiple forms of DNA damage and that there is a general mechanism for generation of the checkpoint-activating signal following DNA damage.

Subfields: Cancer Research, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/6c494949dca9071b729493e8a4d0fd677dcd7a75

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Citations in the corpus

The DNA Damage Response: Making it safe to play with knives (2010, background): “RPA: Sensor of DNA Replication Stress Bulky DNA lesions can lead to arrest of leading strand synthesis at the replication fork and formation of extensive RPA-coated ssDNA regions due to the uncoupling between the MCM helicase and the DNA polymerase (Figure 1B) (Byun et al., 2005).”
PARP is activated at stalled forks to mediate Mre11‐dependent replication restart and recombination (2009, background): “…may represent the uncoupling of replicative polymerase and heli- case, which occurs at stalled forks and also generates ssDNA (Walter and Newport, 2000; Byun et al, 2005), these may be longer stretches of ssDNA, which we show do not activate PARP and may, therefore, be sensed in some other way.”
Continued primer synthesis at stalled replication forks contributes to checkpoint activation (2010, methodology): “To determine how the appearance of these products is related to other events that occur upon replication fork stalling, such as checkpoint activation and helicase–polymerase uncoupling, plasmid unwinding and Chk1 phosphorylation were analyzed in parallel (Walter and Newport, 2000; Byun et al., 2005)”
DNA damage-induced cell death: from specific DNA lesions to the DNA damage response and apoptosis. (2013, background): “Under replication blocking conditions these long stretches of RPAbound single-stranded DNA [98] persist and the serine/threonine protein kinase ATR is recruited to these sites by ATRIP (ATR-interacting protein) [98].”
The CMG helicase bypasses DNA-protein cross-links to facilitate their repair (2018, neutral): “CMG Slows Down after Uncoupling from the Leading Strand At sites of leading strand DNA damage or when DNA synthesis is inhibited, CMG uncouples from the point of synthesis, leading to ssDNA generation and ATR checkpoint activation (Byun et al., 2005; Taylor and Yeeles, 2018).”
The DNA damage response: ten years after. (2007, background): “protein complex RPA, and this unwinding is required for signaling (Byun et al., 2005).”
DNA damage sensing by the ATM and ATR kinases. (2013, background): “Furthermore, increasing amounts of RPA-ssDNA is generated at or behind replication forks when the coordination between helicase and DNA polymerases is compromised by DNA damage and other impediments (Byun et al. 2005; Lopes et al. 2006).”
ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response. (2017, background): “, 1999), or by helicase-polymerase uncoupling at stalled replication forks (Byun et al., 2005).”