DNA replication defects, spontaneous DNA damage, and ATM-dependent checkpoint activation in replication protein A-deficient cells.
Replication protein A (RPA) is a heterotrimeric, single-stranded DNA-binding complex comprised of 70-kDa (RPA1), 32-kDa (RPA2), and 14-kDa (RPA3) subunits that is essential for DNA replication, recombination, and repair in eukaryotes. In addition, recent studies using vertebrate model systems have suggested an important role for RPA in the initiation of cell cycle checkpoints following exposure to DNA replication stress. Specifically, RPA has been implicated in the recruitment and activation of the ATM-Rad3-related protein kinase, ATR, which in conjunction with the related kinase, ATM (ataxia-telangiectasia-mutated), transmits checkpoint signals via the phosphorylation of downstream effectors. In this report, we have explored the effects of RPA insufficiency on DNA replication, cell survival, and ATM/ATR-dependent signal transduction in response to genotoxic stress. RNA interference-mediated suppression of RPA1 caused a slowing of S phase progression, G2/M cell cycle arrest, and apoptosis in HeLa cells. RPA-deficient cells demonstrated high levels of spontaneous DNA damage and constitutive activation of ATM, which was responsible for the terminal G2/M arrest phenotype. Surprisingly, we found that neither RPA1 nor RPA2 were essential for the hydroxyurea- or UV-induced phosphorylation of the ATR substrates CHK1 and CREB (cyclic AMP-response element-binding protein). These findings reveal that RPA is required for genomic stability and suggest that activation of ATR can occur through RPA-independent pathways.
Dodson
Gerald E
GE
Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Shi
Yuling
Y
Tibbetts
Randal S
RS
D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D004249:DNA Damage / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000172:deficiency* / Q000235:genetics / Q000502:physiology; D016195:G2 Phase; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008938:Mitosis; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D051716:Replication Protein A; D012694:Serine / Q000378:metabolism; D014162:Transfection; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; C497167:RPA1 protein, human; D051716:Replication Protein A; D025521:Tumor Suppressor Proteins; D012694:Serine; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases