Abstract
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.
Highlights
Replication protein A (RPA) is a trimeric complex composed of 70 kDa (RPA1), 32 kDa (RPA2), and 14 kDa (RPA3) subunits that is essential for DNA replication in all organisms [1]
Immunoblotting confirmed that the expression of RPA1 was reduced by more than 90% in both cell lines within 48 h of transfection, whereas the expression of RPA2 was unaffected (Fig. 1A)
Transfection of a scrambled (SCR) smallinterfering RNA (siRNA) had no effect on RPA1 expression
Summary
Replication protein A (RPA) is a heterotrimeric, single-stranded DNA (ssDNA)-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. We have explored the effects of RPA insufficiency on DNA replication, cell survival, and ATM/ATRdependent signal transduction in response to genotoxic stress. 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. We found that neither RPA1 nor RPA2 were essential for the HU- or UV-induced phosphorylation of the ATR substrates CHK1 and CREB. These findings reveal that RPA is required for genomic stability and suggest that activation of ATR can occur through RPA-independent pathways in response to genotoxic stress
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