TopBP1 and DNA polymerase-alpha directly recruit the 9-1-1 complex to stalled DNA replication forks.
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.
Yan
Shan
S
The Biological Laboratories, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
Michael
W Matthew
WM
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000378:metabolism; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction; D013312:Stress, Physiological; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
C495984:ATRIP protein, Xenopus; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C511264:Hus1 protein, Xenopus; D046912:Multiprotein Complexes; C511263:Rad1 protein, Xenopus; C509993:TopBP1 protein, Xenopus; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; C069930:rad9 protein; D004247:DNA; C576898:Atr protein, Xenopus; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D004256:DNA Polymerase I