Elsevier

Experimental Cell Research

Volume 300, Issue 2, 1 November 2004, Pages 320-334
Experimental Cell Research

Replication protein A and γ-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks

https://doi.org/10.1016/j.yexcr.2004.07.022Get rights and content

Abstract

Human replication protein A (RPA p34), a crucial component of diverse DNA excision repair pathways, is implicated in DNA double-strand break (DSB) repair. To evaluate its role in DSB repair, the intranuclear dynamics of RPA was investigated after DNA damage and replication blockage in human cells. Using two different agents [ionizing radiation (IR) and hydroxyurea (HU)] to generate DSBs, we found that RPA relocated into distinct nuclear foci and colocalized with a well-known DSB binding factor, γ-H2AX, at the sites of DNA damage in a time-dependent manner. Colocalization of RPA and γ-H2AX foci peaked at 2 h after IR treatment and subsequently declined with increasing postrecovery times. The time course of RPA and γ-H2AX foci association correlated well with the DSB repair activity detected by a neutral comet assay. A phosphatidylinositol-3 (PI-3) kinase inhibitor, wortmannin, completely abolished both RPA and γ-H2AX foci formation triggered by IR. Additionally, radiosensitive ataxia telangiectasia (AT) cells harboring mutations in ATM gene product were found to be deficient in RPA and γ-H2AX colocalization after IR. Transfection of AT cells with ATM cDNA fully restored the association of RPA foci with γ-H2AX illustrating the requirement of ATM gene product for this process. The exact coincidence of RPA and γ-H2AX in response to HU specifically in S-phase cells supports their role in DNA replication checkpoint control. Depletion of RPA by small interfering RNA (SiRNA) substantially elevated the frequencies of IR-induced micronuclei (MN) and apoptosis in human cells suggestive of a role for RPA in DSB repair. We propose that RPA in association with γ-H2AX contributes to both DNA damage checkpoint control and repair in response to strand breaks and stalled replication forks in human cells.

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).

References (45)

  • S. Burma et al.

    ATM phosphorylates histone H2AX in response to DNA double-strand breaks

    J. Biol. Chem.

    (2001)
  • I.M. Ward et al.

    Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress

    J. Biol. Chem.

    (2001)
  • T.T. Paull et al.

    A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage

    Curr. Biol.

    (2000)
  • J.S. Liu et al.

    Comparison of checkpoint response triggered by DNA polymerase inhibition versus DNA damaging agents

    Mutat. Res.

    (2003)
  • Y. Andegeko et al.

    Nuclear retention of ATM at sites of DNA double strand breaks

    J. Biol. Chem.

    (2001)
  • C. Iftode et al.

    Replication protein A (RPA): the eukaryotic SSB

    Crit. Rev. Biochem. Mol. Biol.

    (1999)
  • Z. He et al.

    RPA involvement in the damage-recognition and incision steps of nucleotide excision repair

    Nature

    (1995)
  • G.L. Dianov et al.

    Replication protein A stimulates proliferating cell nuclear antigen-dependent repair of abasic sites in DNA by human cell extracts

    Biochemistry

    (1999)
  • M.S. Park et al.

    Physical interaction between human RAD52 and RPA is required for homologous recombination in mammalian cells.

    J. Biol. Chem.

    (1996)
  • R.G. Shao et al.

    Replication mediated DNA damage by camptothecin induces phosphorylation of RPA by DNA-dependent protein kinase and dissociates RPA: DNA-PK complexes

    EMBO J.

    (1999)
  • H. Wang et al.

    Replication protein A2 phosphorylation after DNA damage by the coordinated action of ataxia telangiectasia-mutated and DNA-dependent protein kinase

    Cancer. Res.

    (2001)
  • M. Gatei et al.

    Role for ATM in DNA damage-induced phosphorylation of BRCA1

    Cancer Res.

    (2000)
  • Cited by (57)

    • Zscan5b Deficiency Impairs DNA Damage Response and Causes Chromosomal Aberrations during Mitosis

      2019, Stem Cell Reports
      Citation Excerpt :

      Zscan5b-deficient irradiated ESCs also displayed a trend to increased frequency of RPA foci. RPA relocates into distinct nuclear foci and colocalizes with γ-H2AX at the sites of DNA damage in a time-dependent manner (Balajee and Geard, 2004). These results suggest that DNA repair processes after the initial DNA damage response do not work in Zscan5b-deficient ESCs.

    • In silico identification of inhibitors targeting N-Terminal domain of human Replication Protein A

      2019, Journal of Molecular Graphics and Modelling
      Citation Excerpt :

      DNA damage response (DDR) is the collective term referring to all of the strategies allocated by cells to deal with the DNA damage that can occur in every cell every day [1–5].

    • Significant impact of divalent metal ions on the fidelity, sugar selectivity, and drug incorporation efficiency of human PrimPol

      2017, DNA Repair
      Citation Excerpt :

      Interestingly, PrimPol is found in both the nucleus and the mitochondria and has been implicated in replication fork progression and restart, as well as DNA lesion bypass [1,3,5,6]. Gene silencing of PrimPol in human cells causes profound arrest in mitochondrial DNA (mtDNA) synthesis, decreased replication fork progression rates, and increased replication protein A (RPA) foci, which is indicative of replicative stress [1,3,5,7]. In PrimPol−/− derived mouse embryonic fibroblasts, the presence of chromosomal aberrations such as chromatid breaks [2,3] and micronuclei [5] indicate that PrimPol is essential in the maintenance of genomic integrity.

    • Radiation-induced bystander and systemic effects serve as a unifying model system for genotoxic stress responses

      2018, Mutation Research - Reviews in Mutation Research
      Citation Excerpt :

      Single stranded DNA at the sites of stalled replication forks attracts replication protein A (RPA) which is induced in bystander cells [85]. RPA co-localises with γH2 AX, a marker for DNA double strand breaks [67], in S-phase cells in an ATM-dependent manner [86]. Secondary DSBs are thought to underlie the formation of subnuclear foci of γH2 AX [42,82,87] and 53BP1 [30,61] in bystander cells.

    View all citing articles on Scopus
    View full text