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:

BLM has early and late functions in homologous recombination repair in mouse embryonic stem cells

Oncogene volume 29, pages 4705–4714 (2010)Cite this article

Subjects

Abstract

BLM is a RecQ family helicase that is defective in individuals with the cancer predisposition disorder, Bloom's syndrome (BS). At the cellular level, BS is characterized by hyper-recombination manifested as excessive sister chromatid exchange and loss of heterozygosity. However, the precise function of BLM remains unclear. Multiple roles have been proposed for BLM in the homologous recombination (HR) repair pathway, including ‘early’ functions, such as the stimulation of resection of DNA double-strand break ends or displacement of the invading strand of DNA displacement loops, and ‘late’ roles, such as dissolution of double Holliday junctions. However, most of the evidence for these putative roles comes from in vitro biochemical data. In this study, we report the characterization of mouse embryonic stem cells with disruption of Blm and/or Rad54 genes. We show that Blm has roles both upstream and downstream of the Rad54 protein, a core HR factor. Disruption of Rad54 in the Blm-mutant background reduced the elevated level of gene targeting and of sister chromatid exchanges, implying that Blm primarily functions downstream of Rad54 in the HR pathway. Conversely, however, mutation of Blm in Rad54−/− cells rescued their mitomycin C (MMC) sensitivity, and decreased both the level of DNA damage and cell cycle perturbation induced by MMC, suggesting an early role for Blm. Our data are consistent with Blm having at least two roles in HR repair in mammalian cells.

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
Figure 6

Similar content being viewed by others

References

  • Amitani I, Baskin RJ, Kowalczykowski SC . (2006). Visualization of Rad54, a chromatin remodeling protein, translocating on single DNA molecules. Mol Cell 23: 143–148.

    Article  CAS  Google Scholar 

  • Bachrati CZ, Borts RH, Hickson ID . (2006). Mobile D-loops are a preferred substrate for the Bloom's syndrome helicase. Nucleic Acids Res 34: 2269–2279.

    Article  CAS  Google Scholar 

  • Beard C, Hochedlinger K, Plath K, Wutz A, Jaenisch R . (2006). Efficient method to generate single-copy transgenic mice by site-specific integration in embryonic stem cells. Genesis 44: 23–28.

    Article  CAS  Google Scholar 

  • Bugreev DV, Mazina OM, Mazin AV . (2006). Rad54 protein promotes branch migration of Holliday junctions. Nature 442: 590–593.

    Article  CAS  Google Scholar 

  • Bugreev DV, Yu X, Egelman EH, Mazin AV . (2007). Novel pro- and anti-recombination activities of the Bloom's syndrome helicase. Genes Dev 21: 3085–3094.

    Article  CAS  Google Scholar 

  • Chaganti RS, Schonberg S, German J . (1974). A manyfold increase in sister chromatid exchanges in Bloom's syndrome lymphocytes. Proc Natl Acad Sci USA 71: 4508–4512.

    Article  CAS  Google Scholar 

  • Chu WK, Hickson ID . (2009). RecQ helicases: multifunctional genome caretakers. Nat Rev Cancer 9: 644–654.

    Article  CAS  Google Scholar 

  • Dillehay LE, Jacobson-Kram D, Williams JR . (1989). DNA topoisomerases and models of sister-chromatid exchange. Mutat Res 215: 15–23.

    Article  CAS  Google Scholar 

  • Dronkert ML, Beverloo HB, Johnson RD, Hoeijmakers JH, Jasin M, Kanaar R . (2000). Mouse RAD54 affects DNA double-strand break repair and sister chromatid exchange. Mol Cell Biol 20: 3147–3156.

    Article  CAS  Google Scholar 

  • Eisen JA, Sweder KS, Hanawalt PC . (1995). Evolution of the SNF2 family of proteins: subfamilies with distinct sequences and functions. Nucleic Acids Res 23: 2715–2723.

    Article  CAS  Google Scholar 

  • Essers J, Hendriks RW, Swagemakers SM, Troelstra C, de Wit J, Bootsma D et al. (1997). Disruption of mouse RAD54 reduces ionizing radiation resistance and homologous recombination. Cell 89: 195–204.

    Article  CAS  Google Scholar 

  • Essers J, Houtsmuller AB, van Veelen L, Paulusma C, Nigg AL, Pastink A et al. (2002). Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage. Embo J 21: 2030–2037.

    Article  CAS  Google Scholar 

  • Gravel S, Chapman JR, Magill C, Jackson SP . (2008). DNA helicases Sgs1 and BLM promote DNA double-strand break resection. Genes Dev 22: 2767–2772.

    Article  CAS  Google Scholar 

  • Hanada K, Budzowska M, Modesti M, Maas A, Wyman C, Essers J et al. (2006). The structure-specific endonuclease Mus81-Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks. Embo J 25: 4921–4932.

    Article  CAS  Google Scholar 

  • Heyer WD, Li X, Rolfsmeier M, Zhang XP . (2006). Rad54: the Swiss Army knife of homologous recombination? Nucleic Acids Res 34: 4115–4125.

    Article  CAS  Google Scholar 

  • Interthal H, Heyer WD . (2000). MUS81 encodes a novel helix-hairpin-helix protein involved in the response to UV- and methylation-induced DNA damage in Saccharomyces cerevisiae. Mol Gen Genet 263: 812–827.

    Article  CAS  Google Scholar 

  • Li X, Stith CM, Burgers PM, Heyer WD . (2009). PCNA is required for initiation of recombination-associated DNA synthesis by DNA polymerase delta. Mol Cell 36: 704–713.

    Article  Google Scholar 

  • Lim DS, Hasty P . (1996). A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. Mol Cell Biol 16: 7133–7143.

    Article  CAS  Google Scholar 

  • Luo G, Santoro IM, McDaniel LD, Nishijima I, Mills M, Youssoufian H et al. (2000). Cancer predisposition caused by elevated mitotic recombination in Bloom mice. Nat Genet 26: 424–429.

    Article  CAS  Google Scholar 

  • Mankouri HW, Ngo HP, Hickson ID . (2007). Shu proteins promote the formation of homologous recombination intermediates that are processed by Sgs1-Rmi1-Top3. Mol Biol Cell 18: 4062–4073.

    Article  CAS  Google Scholar 

  • Marple T, Kim TM, Hasty P . (2006). Embryonic stem cells deficient for Brca2 or Blm exhibit divergent genotoxic profiles that support opposing activities during homologous recombination. Mutat Res 602: 110–120.

    Article  CAS  Google Scholar 

  • Mazina OM, Mazin AV . (2008). Human Rad54 protein stimulates human Mus81-Eme1 endonuclease. Proc Natl Acad Sci USA 105: 18249–18254.

    Article  CAS  Google Scholar 

  • Mimitou EP, Symington LS . (2008). Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing. Nature 455: 770–774.

    Article  CAS  Google Scholar 

  • Nimonkar AV, Ozsoy AZ, Genschel J, Modrich P, Kowalczykowski SC . (2008). Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair. Proc Natl Acad Sci USA 105: 16906–16911.

    Article  CAS  Google Scholar 

  • Otsuki M, Seki M, Inoue E, Yoshimura A, Kato G, Yamanouchi S et al. (2007). Functional interactions between BLM and XRCC3 in the cell. J Cell Biol 179: 53–63.

    Article  CAS  Google Scholar 

  • Ralf C, Hickson ID, Wu L . (2006). The Bloom's syndrome helicase can promote the regression of a model replication fork. J Biol Chem 281: 22839–22846.

    Article  CAS  Google Scholar 

  • Raynard S, Bussen W, Sung P . (2006). A double Holliday junction dissolvasome comprising BLM, topoisomerase IIIalpha, and BLAP75. J Biol Chem 281: 13861–13864.

    Article  CAS  Google Scholar 

  • Rijkers T, Van Den Ouweland J, Morolli B, Rolink AG, Baarends WM, Van Sloun PP et al. (1998). Targeted inactivation of mouse RAD52 reduces homologous recombination but not resistance to ionizing radiation. Mol Cell Biol 18: 6423–6429.

    Article  CAS  Google Scholar 

  • Ristic D, Wyman C, Paulusma C, Kanaar R . (2001). The architecture of the human Rad54-DNA complex provides evidence for protein translocation along DNA. Proc Natl Acad Sci USA 98: 8454–8460.

    Article  CAS  Google Scholar 

  • Singh TR, Ali AM, Busygina V, Raynard S, Fan Q, Du CH et al. (2008). BLAP18/RMI2, a novel OB-fold-containing protein, is an essential component of the bloom helicase-double Holliday junction dissolvasome. Genes Dev 22: 2856–2868.

    Article  CAS  Google Scholar 

  • Solinger JA, Kiianitsa K, Heyer WD . (2002). Rad54, a Swi2/Snf2-like recombinational repair protein, disassembles Rad51:dsDNA filaments. Mol Cell 10: 1175–1188.

    Article  CAS  Google Scholar 

  • Sonoda E, Sasaki MS, Morrison C, Yamaguchi-Iwai Y, Takata M, Takeda S . (1999). Sister chromatid exchanges are mediated by homologous recombination in vertebrate cells. Mol Cell Biol 19: 5166–5169.

    Article  CAS  Google Scholar 

  • Swagemakers SM, Essers J, de Wit J, Hoeijmakers JH, Kanaar R . (1998). The human RAD54 recombinational DNA repair protein is a double-stranded DNA-dependent ATPase. J Biol Chem 273: 28292–28297.

    Article  CAS  Google Scholar 

  • Tan TL, Kanaar R, Wyman C . (2003). Rad54, a Jack of all trades in homologous recombination. DNA Repair (Amst) 2: 787–794.

    Article  Google Scholar 

  • te Riele H, Maandag ER, Berns A . (1992). Highly efficient gene targeting in embryonic stem cells through homologous recombination with isogenic DNA constructs. Proc Natl Acad Sci USA 89: 5128–5132.

    Article  CAS  Google Scholar 

  • Tsuzuki T, Fujii Y, Sakumi K, Tominaga Y, Nakao K, Sekiguchi M et al. (1996). Targeted disruption of the Rad51 gene leads to lethality in embryonic mice. Proc Natl Acad Sci USA 93: 6236–6240.

    Article  CAS  Google Scholar 

  • van Brabant AJ, Ye T, Sanz M, German IJ, Ellis NA, Holloman WK . (2000). Binding and melting of D-loops by the Bloom syndrome helicase. Biochemistry 39: 14617–14625.

    Article  CAS  Google Scholar 

  • Van Komen S, Petukhova G, Sigurdsson S, Stratton S, Sung P . (2000). Superhelicity-driven homologous DNA pairing by yeast recombination factors Rad51 and Rad54. Mol Cell 6: 563–572.

    Article  CAS  Google Scholar 

  • Wang W, Seki M, Narita Y, Sonoda E, Takeda S, Yamada K et al. (2000). Possible association of BLM in decreasing DNA double strand breaks during DNA replication. Embo J 19: 3428–3435.

    Article  CAS  Google Scholar 

  • Wesoly J, Agarwal S, Sigurdsson S, Bussen W, Van Komen S, Qin J et al. (2006). Differential contributions of mammalian Rad54 paralogs to recombination, DNA damage repair, and meiosis. Mol Cell Biol 26: 976–989.

    Article  CAS  Google Scholar 

  • Wu L, Bachrati CZ, Ou J, Xu C, Yin J, Chang M et al. (2006). BLAP75/RMI1 promotes the BLM-dependent dissolution of homologous recombination intermediates. Proc Natl Acad Sci USA 103: 4068–4073.

    Article  CAS  Google Scholar 

  • Wu L, Davies SL, Levitt NC, Hickson ID . (2001). Potential role for the BLM helicase in recombinational repair via a conserved interaction with RAD51. J Biol Chem 276: 19375–19381.

    Article  CAS  Google Scholar 

  • Wu L, Hickson ID . (2003). The Bloom's syndrome helicase suppresses crossing over during homologous recombination. Nature 426: 870–874.

    Article  CAS  Google Scholar 

  • Xu D, Guo R, Sobeck A, Bachrati CZ, Yang J, Enomoto T et al. (2008). RMI, a new OB-fold complex essential for Bloom syndrome protein to maintain genome stability. Genes Dev 22: 2843–2855.

    Article  CAS  Google Scholar 

  • Zhang R, Sengupta S, Yang Q, Linke SP, Yanaihara N, Bradsher J et al. (2005). BLM helicase facilitates Mus81 endonuclease activity in human cells. Cancer Res 65: 2526–2531.

    Article  CAS  Google Scholar 

  • Zhu Z, Chung WH, Shim EY, Lee SE, Ira G . (2008). Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends. Cell 134: 981–994.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Dr A Bradley for Blmm3/m4 cells, and Drs H te Riele and D Adams for the targeting vectors. We also thank Drs L Wu, P McHugh and CZ Bachrati for helpful comments on the paper, Mr PS North for advice on SCE assays and Miss P White for preparing the paper. This work was supported by Cancer Research UK and by The Bloom's Syndrome Foundation (IDH), and by The Netherlands Genomic Initiative/The Netherlands Organization for Scientific Research (RK). WK Chu was supported by the Anita Mui ‘True Heart’ Charity Foundation, the K P Tin Foundation Limited and the China Oxford Scholarship Fund.

Author information

Authors and Affiliations

  1. Weatherall Institute of Molecular Medicine, John Radcliffe Hospital,

    W K Chu, K Hanada & I D Hickson

  2. University of Oxford, Oxford, UK

    W K Chu, K Hanada & I D Hickson

  3. Department of Cell Biology and Genetics, Department of Radiation Oncology, Cancer Genomics Center, Erasmus MC, Rotterdam, The Netherlands

    K Hanada & R Kanaar

  4. Department of Cellular and Molecular Medicine, Panum Institute, University of Copenhagen, Copenhagen N, Denmark

    I D Hickson

Authors
  1. W K Chu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. K Hanada
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R Kanaar
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. I D Hickson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to I D Hickson.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chu, W., Hanada, K., Kanaar, R. et al. BLM has early and late functions in homologous recombination repair in mouse embryonic stem cells. Oncogene 29, 4705–4714 (2010). https://doi.org/10.1038/onc.2010.214

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2010.214

Keywords

This article is cited by

Search

Advanced search

Quick links