Skip to main content
SpringerLink
Log in

Cellular Assays to Study the Functional Importance of Human DNA Repair Helicases

  • Protocol
  • First Online:
DNA Repair

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1999))

Abstract

DNA helicases represent a specialized class of enzymes that play crucial roles in the DNA damage response. Using the energy of nucleoside triphosphate binding and hydrolysis, helicases behave as molecular motors capable of efficiently disrupting the many noncovalent hydrogen bonds that stabilize DNA molecules with secondary structure. In addition to their importance in DNA damage sensing and signaling, DNA helicases facilitate specific steps in DNA repair mechanisms that require polynucleotide tract unwinding or resolution. Because they play fundamental roles in the DNA damage response and DNA repair, defects in helicases disrupt cellular homeostasis. Thus, helicase deficiency or inhibition may result in reduced cell proliferation and survival, apoptosis, DNA damage induction, defective localization of repair proteins to sites of genomic DNA damage, chromosomal instability, and defective DNA repair pathways such as homologous recombination of double-strand breaks. In this chapter, we will describe step-by-step protocols to assay the functional importance of human DNA repair helicases in genome stability and cellular homeostasis.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Estep KN, Brosh RM Jr (2018) RecQ and Fe-S helicases have unique roles in DNA metabolism dictated by their unwinding directionality, substrate specificity, and protein interactions. Biochem Soc Trans 46:77–95. https://doi.org/10.1042/bst20170044

    Article  CAS  PubMed  Google Scholar 

  2. Suhasini AN, Brosh RM Jr (2013) Disease-causing missense mutations in human DNA helicase disorders. Mutat Res 752(2):138–152. https://doi.org/10.1016/j.mrrev.2012.12.004

    Article  CAS  PubMed  Google Scholar 

  3. van Brabant AJ, Stan R, Ellis NA (2000) DNA helicases, genomic instability, and human genetic disease. Annu Rev Genomics Hum Genet 1:409–459. https://doi.org/10.1146/annurev.genom.1.1.409

    Article  PubMed  Google Scholar 

  4. Byrd AK, Raney KD (2012) Superfamily 2 helicases. Front Biosci (Landmark Ed) 17:2070–2088

    Article  Google Scholar 

  5. Gilman B, Tijerina P, Russell R (2017) Distinct RNA-unwinding mechanisms of DEAD-box and DEAH-box RNA helicase proteins in remodeling structured RNAs and RNPs. Biochem Soc Trans 45(6):1313–1321. https://doi.org/10.1042/bst20170095

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Raney KD, Byrd AK, Aarattuthodiyil S (2013) Structure and mechanisms of SF1 DNA helicases. Adv Exp Med Biol 767:17–46. https://doi.org/10.1007/978-1-4614-5037-5_2

    Article  CAS  PubMed  Google Scholar 

  7. Trakselis MA (2016) Structural mechanisms of hexameric helicase loading, assembly, and unwinding. F1000Res 5. F1000 Faculty Rev-111. https://doi.org/10.12688/f1000research.7509.1

    Article  Google Scholar 

  8. Brosh RM Jr, Majumdar A, Desai S, Hickson ID, Bohr VA, Seidman MM (2001) Unwinding of a DNA triple helix by the Werner and Bloom syndrome helicases. J Biol Chem 276(5):3024–3030. https://doi.org/10.1074/jbc.M006784200

    Article  CAS  PubMed  Google Scholar 

  9. Guo M, Hundseth K, Ding H, Vidhyasagar V, Inoue A, Nguyen CH, Zain R, Lee JS, Wu Y (2015) A distinct triplex DNA unwinding activity of ChlR1 helicase. J Biol Chem 290(8):5174–5189. https://doi.org/10.1074/jbc.M114.634923

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Mohaghegh P, Karow JK, Brosh RM Jr, Bohr VA, Hickson ID (2001) The Bloom’s and Werner’s syndrome proteins are DNA structure-specific helicases. Nucleic Acids Res 29(13):2843–2849

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Sun H, Karow JK, Hickson ID, Maizels N (1998) The Bloom’s syndrome helicase unwinds G4 DNA. J Biol Chem 273(42):27587–27592

    Article  CAS  PubMed  Google Scholar 

  12. Vaughn JP, Creacy SD, Routh ED, Joyner-Butt C, Jenkins GS, Pauli S, Nagamine Y, Akman SA (2005) The DEXH protein product of the DHX36 gene is the major source of tetramolecular quadruplex G4-DNA resolving activity in HeLa cell lysates. J Biol Chem 280(46):38117–38120. https://doi.org/10.1074/jbc.C500348200

    Article  CAS  PubMed  Google Scholar 

  13. Bacolla A, Wang G, Jain A, Chuzhanova NA, Cer RZ, Collins JR, Cooper DN, Bohr VA, Vasquez KM (2011) Non-B DNA-forming sequences and WRN deficiency independently increase the frequency of base substitution in human cells. J Biol Chem 286(12):10017–10026. https://doi.org/10.1074/jbc.M110.176636

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sharma S, Doherty Kevin M, Brosh Robert M (2006) Mechanisms of RecQ helicases in pathways of DNA metabolism and maintenance of genomic stability. Biochem J 398(Pt 3):319–337. https://doi.org/10.1042/BJ20060450

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wu Y (2012) Unwinding and rewinding: double faces of helicase? J Nucl Acids 2012:140601. https://doi.org/10.1155/2012/140601

    Article  CAS  Google Scholar 

  16. Brosh RM (2013) DNA helicases involved in DNA repair and their roles in cancer. Nat Rev Cancer 13(8):542–558. https://doi.org/10.1038/nrc3560

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sharma S (2014) An appraisal of RECQ1 expression in cancer progression. Front Genet 5:426. https://doi.org/10.3389/fgene.2014.00426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Arai A, Chano T, Futami K, Furuichi Y, Ikebuchi K, Inui T, Tameno H, Ochi Y, Shimada T, Hisa Y, Okabe H (2011) RECQL1 and WRN proteins are potential therapeutic targets in head and neck squamous cell carcinoma. Cancer Res 71(13):4598–4607. https://doi.org/10.1158/0008-5472.can-11-0320

    Article  CAS  PubMed  Google Scholar 

  19. Sharma S, Brosh RM Jr (2007) Human RECQ1 is a DNA damage responsive protein required for genotoxic stress resistance and suppression of sister chromatid exchanges. PLoS One 2(12):e1297. https://doi.org/10.1371/journal.pone.0001297

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Aggarwal M, Banerjee T, Sommers JA, Brosh RM Jr (2013) Targeting an Achilles’ heel of cancer with a WRN helicase inhibitor. Cell Cycle 12(20):3329–3335. https://doi.org/10.4161/cc.26320

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Aggarwal M, Banerjee T, Sommers JA, Iannascoli C, Pichierri P, Shoemaker RH, Brosh RM Jr (2013) Werner syndrome helicase has a critical role in DNA damage responses in the absence of a functional Fanconi anemia pathway. Cancer Res 73(17):5497–5507. https://doi.org/10.1158/0008-5472.can-12-2975

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Aggarwal M, Sommers JA, Shoemaker RH, Brosh RM Jr (2011) Inhibition of helicase activity by a small molecule impairs Werner syndrome helicase (WRN) function in the cellular response to DNA damage or replication stress. Proc Natl Acad Sci U S A 108(4):1525–1530. https://doi.org/10.1073/pnas.1006423108

    Article  PubMed  PubMed Central  Google Scholar 

  23. Nguyen GH, Dexheimer TS, Rosenthal AS, Chu WK, Singh DK, Mosedale G, Bachrati CZ, Schultz L, Sakurai M, Savitsky P, Abu M, McHugh PJ, Bohr VA, Harris CC, Jadhav A, Gileadi O, Maloney DJ, Simeonov A, Hickson ID (2013) A small molecule inhibitor of the BLM helicase modulates chromosome stability in human cells. Chem Biol 20(1):55–62. https://doi.org/10.1016/j.chembiol.2012.10.016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Estep KN, Butler TJ, Ding J, Brosh RM Jr (2017) G4-interacting DNA helicases and polymerases: potential therapeutic targets. Curr Med Chem. https://doi.org/10.2174/0929867324666171116123345

  25. Hengel SR, Spies MA, Spies M (2017) Small-molecule inhibitors targeting DNA repair and DNA repair deficiency in research and cancer therapy. Cell Chem Biol 24(9):1101–1119. https://doi.org/10.1016/j.chembiol.2017.08.027

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Brosh RM Jr, Bohr VA (2007) Human premature aging, DNA repair and RecQ helicases. Nucleic Acids Res 35(22):7527–7544. https://doi.org/10.1093/nar/gkm1008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pierce AJ, Johnson RD, Thompson LH, Jasin M (1999) XRCC3 promotes homology-directed repair of DNA damage in mammalian cells. Genes Dev 13(20):2633–2638

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Berridge MV, Herst PM, Tan AS (2005) Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev 11:127–152. https://doi.org/10.1016/s1387-2656(05)11004-7

    Article  CAS  PubMed  Google Scholar 

  29. Wu Y, Shin-ya K, Brosh RM Jr (2008) FANCJ helicase defective in Fanconia anemia and breast cancer unwinds G-quadruplex DNA to defend genomic stability. Mol Cell Biol 28(12):4116–4128. https://doi.org/10.1128/mcb.02210-07

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Khodarev NN, Sokolova IA, Vaughan AT (1998) Mechanisms of induction of apoptotic DNA fragmentation. Int J Radiat Biol 73(5):455–467

    Article  CAS  PubMed  Google Scholar 

  31. Bennett BT, Bewersdorf J, Knight KL (2009) Immunofluorescence imaging of DNA damage response proteins: optimizing protocols for super-resolution microscopy. Methods 48(1):63–71. https://doi.org/10.1016/j.ymeth.2009.02.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Awasthi P, Foiani M, Kumar A (2015) ATM and ATR signaling at a glance. J Cell Sci 128(23):4255–4262. https://doi.org/10.1242/jcs.169730

    Article  CAS  PubMed  Google Scholar 

  33. Mirzoeva OK, Petrini JH (2001) DNA damage-dependent nuclear dynamics of the Mre11 complex. Mol Cell Biol 21(1):281–288. https://doi.org/10.1128/MCB.21.1.281-288.2001

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Suhasini AN, Sommers JA, Muniandy PA, Coulombe Y, Cantor SB, Masson JY, Seidman MM, Brosh RM Jr (2013) Fanconi anemia group J helicase and MRE11 nuclease interact to facilitate the DNA damage response. Mol Cell Biol 33(11):2212–2227. https://doi.org/10.1128/mcb.01256-12

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Litman R, Peng M, Jin Z, Zhang F, Zhang J, Powell S, Andreassen PR, Cantor SB (2005) BACH1 is critical for homologous recombination and appears to be the Fanconi anemia gene product FANCJ. Cancer Cell 8(3):255–265. https://doi.org/10.1016/j.ccr.2005.08.004

    Article  CAS  PubMed  Google Scholar 

  36. Bharti SK, Sommers JA, George F, Kuper J, Hamon F, Shin-ya K, Teulade-Fichou MP, Kisker C, Brosh RM Jr (2013) Specialization among iron-sulfur cluster helicases to resolve G-quadruplex DNA structures that threaten genomic stability. J Biol Chem 288:28217–28229

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Henderson A, Wu Y, Huang YC, Chavez EA, Platt J, Johnson FB, Brosh RM Jr, Sen D, Lansdorp PM (2014) Detection of G-quadruplex DNA in mammalian cells. Nucleic Acids Res 42:860–869

    Article  CAS  PubMed  Google Scholar 

  38. Wu Y, Sommers JA, Suhasini AN, Leonard T, Deakyne JS, Mazin AV, Shin-Ya K, Kitao H, Brosh RM Jr (2010) Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes. Blood 116:3780–3791

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Banerjee T, Sommers JA, Huang J, Seidman MM, Brosh RM Jr (2015) Catalytic strand separation by RECQ1 is required for RPA-mediated response to replication stress. Curr Biol 25:2830–2838

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Constantinou A, Tarsounas M, Karow JK, Brosh RM, Bohr VA, Hickson ID, West SC (2000) Werner’s syndrome protein (WRN) migrates Holliday junctions and co-localizes with RPA upon replication arrest. EMBO Rep 1:80–84

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Sharma S, Otterlei M, Sommers JA, Driscoll HC, Dianov GL, Kao HI, Bambara RA, Brosh RM Jr (2004) WRN helicase and FEN-1 form a complex upon replication arrest and together process branchmigrating DNA structures associated with the replication fork. Mol Biol Cell 15:734–750

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Suhasini AN, Rawtani NA, Wu Y, Sommers JA, Sharma S, Mosedale G, North PS, Cantor SB, Hickson ID, Brosh RM Jr (2011) Interaction between the helicases genetically linked to Fanconi anemia group J and Bloom’s syndrome. EMBO J 30:692–705

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Gupta R, Sharma S, Sommers JA, Kenny MK, Cantor SB, Brosh RM Jr (2007) FANCJ (BACH1) helicase forms DNA damage inducible foci with replication protein A and interacts physically and functionally with the single-stranded DNA-binding protein. Blood 110:2390–2398

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work is supported by the National Institutes of Health, National Institute on Aging.

Author information

Authors and Affiliations

  1. Laboratory of Molecular Gerontology, National Institute on Aging, NIH, NIH Biomedical Research Center, Baltimore, MD, USA

    Sanket Awate, Srijita Dhar, Joshua A. Sommers & Robert M. Brosh Jr.

Authors
  1. Sanket Awate
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Srijita Dhar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joshua A. Sommers
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert M. Brosh Jr.
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Robert M. Brosh Jr. .

Editor information

Editors and Affiliations

  1. Indiana University – Purdue University, Indianapolis, IN, USA

    Lata Balakrishnan

  2. Department of Biological Sciences, University of South Carolina, Columbia, SC, USA

    Jason A. Stewart

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Awate, S., Dhar, S., Sommers, J.A., Brosh, R.M. (2019). Cellular Assays to Study the Functional Importance of Human DNA Repair Helicases. In: Balakrishnan, L., Stewart, J. (eds) DNA Repair. Methods in Molecular Biology, vol 1999. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9500-4_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9500-4_11

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9499-1

  • Online ISBN: 978-1-4939-9500-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation