TopBP1 and DNA polymerase alpha-mediated recruitment of the 9-1-1 complex to stalled replication forks: Implications for a replication restart-based mechanism for ATR checkpoint activation

Abstract

Here, we summarize recent advances in understanding the multiple roles played by TopBP1 in ATR activation at stalled replication forks. We review recent studies showing that TopBP1 controls the loading of 9-1-1 onto stalled replication forks via a pathway that also requires DNA polymerase alpha (pol α). Based on these recent studies, we present a revised model for ATR activation, and speculate that TopBP1-mediated recruitment of pol α and 9-1-1 may couple checkpoint activation to replication restart, when DNA synthesis is blocked on the leading strand of a replication fork. Lastly, we present a new experiment that examines how TopBP1 binds to stalled replication forks, and we identify important new questions that our recent studies have raised regarding how stalled replication forks are sensed by the ATR checkpoint pathway.

Depletion of RAD17 sensitizes pancreatic cancer cells to gemcitabine

“…The first step in recognition of the stalled fork is the recruitment of ataxia telangiectasia and Rad3-related protein (ATR) by binding of its close partner ATR-interacting protein (ATRIP) to replication protein A (RPA) bound to exposed singlestrand DNA (Cortez et al, 2001;Zou and Elledge, 2003). The second step of activating the ATR-CHK1 signalling cascade starts with the recruitment of polymerase a (Pol a) to the leading strand by topoisomerase-2 binding protein 1 (TopBP1), which also binds to RPA (Yan and Michael, 2009). Pol a then creates primer-template junctions on the leading strand and mediates recruitment of the heterotrimeric DNA clamp complex of RAD9-HUS1-RAD1 (9-1-1) (Yan and Michael, 2009).…”

Replication Fork Stalling and Checkpoint Activation by a PKD1 Locus Mirror Repeat Polypurine-Polypyrimidine (Pu-Py) Tract

“…Under these conditions, the ATR and Rad9-Rad1-Hus1 complex proteins are recruited to RPA-bound single-stranded DNA generated by fork opening in the absence of DNA synthesis (72)(73)(74). To investigate whether replication stalling by the IVS21 Pu-Py sequence correlated with checkpoint protein binding, cross-linked chromatin was immunoprecipitated with antibodies against the ATR, RPA70, or Rad9 proteins.…”

TopBP1-mediated DNA processing during mitosis

“…36,37 Somewhat contradictory, TopBP1 has been shown to localize to stalled replication forks independently of the 9-1-1 complex, and rather be responsible for recruitment of 9-1-1. 38,39 In line with this, recruitment of TopBP1 to ATR activating DNA structures in Xenopus extracts was shown to rely on the MRN complex. The MRN-mediated recruitment seemed to depend on TopBP1 BRCT3-6.…”