Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

PC4 promotes genome stability and DNA repair through binding of ssDNA at DNA damage sites

Oncogene volume 35, pages 761–770 (2016)Cite this article

Subjects

Abstract

The transcriptional cofactor PC4 is an ancient single-strand DNA (ssDNA)-binding protein that has a homologue in bacteriophage T5 where it is likely the elusive replicative ssDNA-binding protein. We hypothesize that human PC4 has retained functions in ssDNA binding to stabilize replication forks and prevent genome instability in mammalian cells. Here we demonstrate that PC4 is recruited to hydroxyurea (HU)-stalled replication forks, which is dependent on active transcription and its ssDNA-binding ability. Interestingly, we demonstrate that ssDNA binding by PC4 is critical to suppress spontaneous DNA damage and promote cellular survival. PC4 accumulation co-localizes with replication protein A (RPA) at stalled forks and is increased upon RPA depletion, demonstrating compensatory functions in ssDNA binding. Depletion of PC4 not only results in defective resolution of HU-induced DNA damage but also significantly reduces homologous recombination repair efficiency. Altogether, our results indicate that PC4 has similar functions to RPA in binding ssDNA to promote genome stability, especially at sites of replication–transcription collisions.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Barlow JH, Faryabi RB, Callen E, Wong N, Malhowski A, Chen HT et al. Identification of early replicating fragile sites that contribute to genome instability. Cell 2013; 152: 620–632.

    Article  CAS  Google Scholar 

  2. Bartkova J, Rezaei N, Liontos M, Karakaidos P, Kletsas D, Issaeva N et al. Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints. Nature 2006; 444: 633–637.

    Article  CAS  Google Scholar 

  3. Di Micco R, Fumagalli M, Cicalese A, Piccinin S, Gasparini P, Luise C et al. Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication. Nature 2006; 444: 638–642.

    Article  CAS  Google Scholar 

  4. Mortusewicz O, Herr P, Helleday T . Early replication fragile sites: where replication-transcription collisions cause genetic instability. EMBO J 2013; 32: 493–495.

    Article  CAS  Google Scholar 

  5. Toledo LI, Altmeyer M, Rask MB, Lukas C, Larsen DH, Povlsen LK et al. ATR prohibits replication catastrophe by preventing global exhaustion of RPA. Cell 2013; 155: 1088–1103.

    Article  CAS  Google Scholar 

  6. Zou L, Elledge SJ . Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 2003; 300: 1542–1548.

    Article  CAS  Google Scholar 

  7. Vassin VM, Anantha RW, Sokolova E, Kanner S, Borowiec JA . Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress. J Cell Sci 2009; 122: 4070–4080.

    Article  CAS  Google Scholar 

  8. Feijoo C, Hall-Jackson C, Wu R, Jenkins D, Leitch J, Gilbert DM et al. Activation of mammalian Chk1 during DNA replication arrest: a role for Chk1 in the intra-S phase checkpoint monitoring replication origin firing. J Cell Biol 2001; 154: 913–923.

    Article  CAS  Google Scholar 

  9. Steigemann B, Schulz A, Werten S . Bacteriophage T5 encodes a homolog of the eukaryotic transcription coactivator PC4 implicated in recombination-dependent DNA replication. J Mol Biol 2013; 425: 4125–4133.

    Article  CAS  Google Scholar 

  10. Batta K, Yokokawa M, Takeyasu K, Kundu TK . Human transcriptional coactivator PC4 stimulates DNA end joining and activates DSB repair activity. J Mol Biol 2009; 385: 788–799.

    Article  CAS  Google Scholar 

  11. Das C, Hizume K, Batta K, Kumar BR, Gadad SS, Ganguly S et al. Transcriptional coactivator PC4, a chromatin-associated protein, induces chromatin condensation. Mol Cell Biol 2006; 26: 8303–8315.

    Article  CAS  Google Scholar 

  12. Ge H, Roeder RG . Purification, cloning, and characterization of a human coactivator, PC4, that mediates transcriptional activation of class II genes. Cell 1994; 78: 513–523.

    Article  CAS  Google Scholar 

  13. Kretzschmar M, Kaiser K, Lottspeich F, Meisterernst M . A novel mediator of class II gene transcription with homology to viral immediate-early transcriptional regulators. Cell 1994; 78: 525–534.

    Article  CAS  Google Scholar 

  14. Mortusewicz O, Roth W, Li N, Cardoso MC, Meisterernst M, Leonhardt H . Recruitment of RNA polymerase II cofactor PC4 to DNA damage sites. J Cell Biol 2008; 183: 769–776.

    Article  CAS  Google Scholar 

  15. Pan ZQ, Ge H, Amin AA, Hurwitz J . Transcription-positive cofactor 4 forms complexes with HSSB (RPA) on single-stranded DNA and influences HSSB-dependent enzymatic synthesis of simian virus 40 DNA. J Biol Chem 1996; 271: 22111–22116.

    Article  CAS  Google Scholar 

  16. Wang JY, Sarker AH, Cooper PK, Volkert MR . The single-strand DNA binding activity of human PC4 prevents mutagenesis and killing by oxidative DNA damage. Mol Cell Biol 2004; 24: 6084–6093.

    Article  CAS  Google Scholar 

  17. Yu L, Volkert MR . Differential requirement for SUB1 in chromosomal and plasmid double-strand DNA break repair. PLoS One 2013; 8: e58015.

    Article  CAS  Google Scholar 

  18. Suzuki K, Yamauchi M, Oka Y, Suzuki M, Yamashita S . Creating localized DNA double-strand breaks with microirradiation. Nat Protoc 2011; 6: 134–139.

    Article  CAS  Google Scholar 

  19. Petermann E, Orta ML, Issaeva N, Schultz N, Helleday T . Hydroxyurea-talled replication forks become progressively inactivated and require two different RAD51-mediated pathways for restart and repair. Mol Cell 2010; 37: 492–502.

    Article  CAS  Google Scholar 

  20. Arnaudeau C, Lundin C, Helleday T . DNA double-strand breaks associated with replication forks are predominantly repaired by homologous recombination involving an exchange mechanism in mammalian cells. J Mol Biol 2001; 307: 1235–1245.

    Article  CAS  Google Scholar 

  21. Lundin C, Schultz N, Arnaudeau C, Mohindra A, Hansen LT, Helleday T . RAD51 is involved in repair of damage associated with DNA replication in mammalian cells. J Mol Biol 2003; 328: 521–535.

    Article  CAS  Google Scholar 

  22. Werten S, Stelzer G, Goppelt A, Langen FM, Gros P, Timmers HT et al. Interaction of PC4 with melted DNA inhibits transcription. EMBO J 1998; 17: 5103–5111.

    Article  CAS  Google Scholar 

  23. Sakaguchi K, Ishibashi T, Uchiyama Y, Iwabata K . The multi-replication protein A (RPA) system—a new perspective. FEBS J 2009; 276: 943–963.

    Article  CAS  Google Scholar 

  24. Brandsen J, Werten S, van der Vliet PC, Meisterernst M, Kroon J, Gros P . C-terminal domain of transcription cofactor PC4 reveals dimeric ssDNA binding site. Nat Struct Biol 1997; 4: 900–903.

    Article  CAS  Google Scholar 

  25. Kaiser K, Stelzer G, Meisterernst M . The coactivator p15 (PC4) initiates transcriptional activation during TFIIA-TFIID-promoter complex formation. EMBO J 1995; 14: 3520–3527.

    Article  CAS  Google Scholar 

  26. Werten S, Moras D . A global transcription cofactor bound to juxtaposed strands of unwound DNA. Nat Struct Mol Biol 2006; 13: 181–182.

    Article  CAS  Google Scholar 

  27. Alabert C, Bukowski-Wills JC, Lee SB, Kustatscher G, Nakamura K, de Lima Alves F et al. Nascent chromatin capture proteomics determines chromatin dynamics during DNA replication and identifies unknown fork components. Nat Cell Biol 2014; 16: 281–293.

    Article  CAS  Google Scholar 

  28. Aymard F, Bugler B, Schmidt CK, Guillou E, Caron P, Briois S et al. Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks. Nat Struct Mol Biol 2014; 21: 366–374.

    Article  CAS  Google Scholar 

  29. Pierce AJ, Johnson RD, Thompson LH, Jasin M . XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev 1999; 13: 2633–2638.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to express our gratitude to Annette Medhurst and Songmin Ying for help with the fibre assay, Guy Kingham for assistance on the DR-GFP assay and to Mick Woodcock for support with the FACS analysis. This project was supported by the Swedish Research Council, Swedish Cancer Society, EMBO LTF (O.M. 451-2010), Dutch Cancer Society (BE), the Torsten and Ragnar Söderberg Foundation, AXA foundation and the Göran Gustafsson Foundation.

Author information

Authors and Affiliations

  1. Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Science for Life Laboratory, Karolinska Institutet, Stockholm, Sweden

    O Mortusewicz, B Evers & T Helleday

Authors
  1. O Mortusewicz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. B Evers
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. T Helleday
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to O Mortusewicz or T Helleday.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies this paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mortusewicz, O., Evers, B. & Helleday, T. PC4 promotes genome stability and DNA repair through binding of ssDNA at DNA damage sites. Oncogene 35, 761–770 (2016). https://doi.org/10.1038/onc.2015.135

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2015.135

This article is cited by

Search

Advanced search

Quick links