Journal of Molecular Biology
RPA Mediates Recombination Repair During Replication Stress and Is Displaced from DNA by Checkpoint Signalling in Human Cells
Introduction
A complex DNA damage response network has been developed to maintain genetic integrity following DNA damage. The coordinated DNA damage response senses lesions, transduces the damage signal and coordinates DNA repair with transcription, replication, the cell cycle and other cellular processes. A single-stranded DNA intermediate commonly forms during DNA replication and repair. Unprotected single-stranded DNA regions are quickly coated with replication protein A (RPA) in eukaryotic cells. Mammalian RPA is a highly conserved multi-unit protein that is also thought to be involved in maintaining genomic stability.1 RPA is composed of three subunits named for their molecular mass (RPA70, RPA32, and RPA14)2 and all subunits are necessary for RPA function.3 RPA co-localises with the MRE11/RAD50/NBS1 (MRN) complex forming repair foci with lesions containing stalled replication forks or single-stranded DNA regions.4 RPA is likely to participate in DNA replication and repair processes through its interaction with other proteins and its strong affinity for single-stranded DNA. It interacts specifically with many proteins including p53,5 XPA,6., 7. ERCC-1/XPF nuclease,8 XPG,9 uracil DNA glycosylase,10 MRE11,11 RAD5212., 13. and RAD51.14 The interactions between RAD51, RAD52 and RPA stimulate homologous recombination.15., 16., 17. In the current yeast model, RPA coats DNA, and also recruits and interacts with RAD52. The RAD52–RPA–single-stranded DNA co-complex then aids the interaction of RAD51 with DNA. This interaction quickly leads to the formation of a RAD51–single-stranded DNA presynaptic complex,18 and the release of RPA and RAD52. The displaced DNA strand is then stabilised by RPA and RAD52.19., 20. RPA was recently shown to mediate the annealing of the displaced DNA strand with RAD52,19 providing an explanation why RPA foci remain visible longer than RAD51 foci in mouse spermatocytes.21
Following DNA damage and replication arrest in mammalian cells RPA has been shown to attract ATRIP and ATR to single-stranded DNA and activates phosphorylation of checkpoint kinase 1 (Chk1).22 Thus, there is a direct link between mammalian RPA with Chk1 DNA damage signalling and RPA appears to act as a sensor molecule signalling single-stranded DNA regions.22 However, others report that activation of Chk1 by ATR–ATRIP is independent of binding to RPA.23 We have previously shown that the cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair,24 providing a link between RPA and homologous recombination repair. Here, we investigated whether the function of RPA in homologous recombination repair is linked with Chk1 signalling and/or has a direct role in homologous recombination. The data presented here show that RPA is required for homologous recombination repair and binds RAD51 and RAD52 after replication stalling. In addition, our data suggest that Chk1 has a separate role in replacing RPA with RAD51 and RAD52 on DNA in order to participate in homologous recombination repair.
Section snippets
Mammalian cells require RPA for homologous recombination repair
It is clear that RPA has central roles in the cell that are linked with its binding to single-stranded DNA, i.e. in replication, transcription, DNA repair, DNA damage signalling and in recombination.25 Here, we depleted RPA70 in the human colorectal carcinoma cell line SW480.SN3 with small interfering RNA (siRNA) and found about 90% depletion of RPA70 protein levels (Figure 1(a)) occurred in all cells (Figure 1(b)).
It has been shown that a RPA70 knockout in mice produces an embryonic lethal
Discussion
We wanted to determine if human RPA is important to initiate homologous recombination repair at stalled replication forks, as this has not been established in human cells. For this study, we used a short RPA siRNA treatment that is sufficient to deplete the RPA protein, but not to trigger cell death or alterations to the cell cycle. We find that RPA siRNA depleted cells fail to assemble RAD51 foci and fail to repair DSBs produced following replication stalling with HU after 24 h, implicating
Drugs and siRNA
Hydroxyurea (Sigma, St Louis, MO) was dissolved in Dulbecco's modified Eagle's medium (DMEM). Aliquots of UCN-01 (from NCI) were prepared in dimethyl sulphoxide (DMSO) and stored at − 20 °C. Cells were transfected with Oligofectamine reagent (Invitrogen, Carlsbad, CA) according to the manufacturers protocol, and the following siRNAs were used: 5′-AACACUCUAUCCUCUUUCAUGTT-3′ (RPA siRNA), the scramble siRNA was purchased from Dharmacon.
Cell cultures
SW480.SN3 cells (a human colon carcinoma cell line stably
Acknowledgements
We thank Mrs Sue Newton for assistance with FACS and Dr Maria Jasin for the pCMV3xnlsI-SceI vector. The Swedish Cancer Society, the Swedish Children's Cancer Foundation, the Swedish Research Council, the Swedish Pain Relief Foundation, the Danish National Research Foundation and the European Commission (integrated project “DNA repair”), the Medical research Council, Cancer Research UK, the Biological and Biotechnological Sciences Research Council and Yorkshire Cancer Research supported this
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Present address: J. Dziegielewski, Radiation Oncology, University of Maryland, Baltimore, MD 21201, USA.