Replication protein A and γ-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks
Introduction
Replication protein A (RPA) plays important roles in diverse DNA metabolic activities such as replication, repair, and recombination [1]. RPA occurs in a heterotrimeric form comprising three subunits with a molecular mass of 70 kDa (p70), 34 kDa (p34), and 14 kDa (p14), respectively. RPA facilitates the DNA unwinding process during replication initiation and elongation and participates in the DNA damage recognition step of nucleotide excision repair pathway [2], [3], [4] through its interaction with XPA (xeroderma pigmentosum complementation group A) and XPG (XP group G) proteins. RPA also enhances the endonuclease activities of XPF-ERCC1 and XPG [5]. RPA is additionally involved in the long patch base excision repair pathway that removes the oxidized base lesions from the genomic DNA [6]. Evidence for the stimulation of DNA ligase I activity by RPA has been recently obtained [7]. RPA catalyzes the homologous pairing and DNA strand exchange steps through its interaction with recombination proteins Rad51 and Rad52 [8].
Among the three subunits, RPA (p34) is a phosphoprotein, which is differentially phosphorylated during the progression of cells from G1 to S-phase, and becomes hyperphosphorylated in response to DNA damage induced by ionizing radiation (IR), ultraviolet radiation (UV), and DNA replication inhibitors such as hydroxyurea (HU) and aphidicolin (APC). RPA phosphorylation by DNA damage is either attenuated or abolished in cells defective in DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) genes [9], [10], indicating their involvement in RPA (p34) phosphorylation. A direct role for DNA-PK in RPA phosphorylation in response to replication mediated DNA damage by topoisomerase I inhibitor, camptothecin, has been demonstrated [9]. ATM kinase is a crucial factor for initiating a cascade of signal transduction pathways in response to IR treatment. Many well-known double-strand break (DSB) repair factors such as Nbs1, Mre11, and BRCA1 are phosphorylated in response to IR by ATM kinase [11], [12], [13]. Colocalization of RPA and ATM kinase has been reported in the synaptonemal complex of meiotic chromosomes suggestive of functional interaction during homologous recombination [14]. The involvement of RPA both in nonhomologous end-joining (NHEJ) and homologous recombination repair (HRR) pathways has also emerged recently. Perrault et al. [15] demonstrated that DSB rejoining by the NHEJ pathway is facilitated by RPA in vitro, while the nuclear foci formation of RPA after IR is modulated by the functional status of BRCA1 [16]. Interaction of RPA with BRCA1, Rad51, and Rad52 proteins attests to a role for RPA in the recombinational repair of DSBs. MacPhail and Olive [17] suggested that the persistence of RPA foci several hours after IR represents sites of irreparable lesions, and this phenomenon might facilitate identification of radiosensitive cells. Nevertheless, the precise participation of RPA in DSB repair is far from clear.
Investigation of the intranuclear dynamics of RPA foci formation in relation to well-known DSB repair factors may enhance our understanding of RPA participation in DSB repair. With this objective in mind, we have undertaken a microscopy-based approach to characterize the RPA foci formation in response to DNA strand breaks generated by IR and HU. To determine whether the focal sites of RPA form at DSB sites, RPA foci formation was analyzed in combination with a well-known DSB binding factor, phosphorylated histone H2AX (γ-H2AX). Using two different agents to generate DSBs, we found that RPA foci colocalized with γ-H2AX in a time-dependent manner after DNA damage. The time course of RPA and γ-H2AX foci association exactly coincided with the DSB repair kinetics detected by a modified neutral comet assay. Absence of RPA and γ-H2AX foci association after IR in radiation-sensitive AT cells suggests a role for ATM gene product in mediating this response. Our findings suggest that DNA damage-dependent recruitment of RPA to γ-H2AX containing focal sites may comprise a critical DNA damage checkpoint control and repair component in human cells.
Section snippets
Cell lines and culture conditions
Human fibroblast cell lines established from normal (MRC5 and GM637H) and AT (GM8391A and GM5849C) individuals were obtained from Coriell Cell Repository, Camden, NJ. Cells were routinely maintained in 2× Eagle minimal essential medium (E-MEM) supplemented with 15% fetal bovine serum, vitamins, essential amino acids, nonessential amino acids, and antibiotics (Gibco BRL). The cultures were maintained at 37°C in a humidified 5% CO2 atmosphere.
Generation of ATM cDNA-transfected cell lines
Dr. Yosef Shiloh (University of Tel Aviv, Israel)
DNA damage-dependent colocalization of RPA and γ-H2AX foci
The time course of intranuclear distribution of RPA in relation to γ-H2AX foci was examined in SV-40-transformed normal human fibroblasts (GM637H) after IR (1 or 10 Gy of γ-rays irradiation) in comparison to unirradiated cells using a combination of antibodies specific for RPA and γ-H2AX. Two distinct patterns of RPA were observed in the nuclei of unirradiated cells directly fixed in acetone–methanol (1:1). A homogenous distribution of small RPA foci ranging from 300 to 350 in number was
Discussion
In this work, we have investigated the intranuclear dynamics of RPA foci formation and its relevance in DSB repair in human cells. To verify whether RPA foci form at the sites of DSBs, RPA foci were investigated in combination with a well-known DSB binding factor, γ-H2AX.Using two different agents to generate DSBs, we demonstrated that RPA foci colocalized with γ-H2AX in a time-dependent fashion in response to DNA strand breaks and stalled replication forks. Abolition of RPA and γ-H2AX foci
Acknowledgments
This work was supported by research grants awarded to C.R.G. from DHHS, NIH (CA 75061, CA 49062 and RR-11623), DOE Office of Science (BER) (DEFG0298ER62687).
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