TopBP1 and DNA polymerase-α directly recruit the 9-1-1 complex to stalled DNA replication forks

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Abstract
TopBP1 and the Rad9-Rad1-Hus1 (9-1-1) complex activate the ataxia telangiectasia mutated and Rad3-related (ATR) protein kinase at stalled replication forks. ATR is recruited to stalled forks through its binding partner, ATR-interacting protein (ATRIP); however, it is unclear how TopBP1 and 9-1-1 are recruited so that they may join ATR-ATRIP and initiate signaling. In this study, we use Xenopus laevis egg extracts to determine the requirements for 9-1-1 loading. We show that TopBP1 is required for the recruitment of both 9-1-1 and DNA polymerase (pol)-alpha to sites of replication stress. Furthermore, we show that pol-alpha is also directly required for Rad9 loading. Our study identifies an assembly pathway, which is controlled by TopBP1 and includes pol-alpha, that mediates the loading of the 9-1-1 complex onto stalled replication forks. These findings clarify early events in the assembly of checkpoint signaling complexes on DNA and identify TopBP1 as a critical sensor of replication stress.