DNA Interstrand Crosslink Repair


Crosslinking agents such as psoralens, nitrogen mustards or cisplatin are bifunctionally acting chemicals that generate a fraction of their adducts as covalent linkages between complementary deoxyribonucleic acid strands. Since many of these agents are of importance in genetic toxicology and cancer therapy, repair of interstrand crosslinks has been studied extensively in bacteria and in lower and higher eukaryotes. The main repair pathway in Escherichia coli involves the sequential action of nucleotide excision repair (NER) and recombinational repair. In eukaryotes, several repair pathways play important roles not only in repair including NER, translesion synthesis and recombination, but also mismatch repair. Relative contributions of the various pathways depend on cell cycle position and agent used. Eukaryotic proteins that specifically enhance resistance to crosslinking agents have been identified (FANC family of proteins, SNM1).

Key Concepts:

  • Chemicals with two or more correctly spaced reactive groups can covalently link opposing DNA strands.

  • Several repair or tolerance pathways such as nucleotide excision repair, recombination and translesion synthesis can work together to overcome such complex damage.

  • Cell cycle stage may determine the choice of repair pathway combinations.

  • A heritable human syndrome with multiple diverse phenotypes (Fanconi anaemia) has been associated with defects in crosslink repair.

  • By integrating in vitro studies and analysis of Fanconi proteins, current models of replication‚Äźdependent crosslink repair assume the creation of double strand breaks at stalled replication forks that are repaired by homologous recombination.

Keywords: DNA repair; crosslinks; excision; bifunctional alkylation; recombination; translesion synthesis

Figure 1.

Examples of ICL structures. (a) Nitrogen mustard, (b) cisplatin and (c) methoxypsoralen. Reproduced with permission from Noll et al. (). © Frontiers in Bioscience.

Figure 2.

Repair of an ICL in E. coli. (a) In the main pathway, first a single‐stranded DNA fragment is removed by NER by cutting 3′ and 5′ of the crosslink. It stays attached to the complementary strand through the crosslink. A gap is formed by the exonuclease activity of Pol I. The single‐stranded gap is filled by RecA‐dependent strand invasion as the initial step of HR and a second round of excision releases a partially double‐stranded fragment containing the crosslink. (b) The minor pathway found in stationary cells also starts with crosslink excision. Then, however, polymerase II (β) performs translesion synthesis and a second round of NER releases the same product as in (a). Reproduced with permission from Dronkert and Kanaar (). © Elsevier.

Figure 3.

DNA crosslink processing in higher eukaryotes in the context of replication. It is assumed that replication fork stalling or collapse leads to origination of a double‐strand break by structure‐specific endonucleases. Considerable redundancy of their activities is expected. This appears to be a precondition for incision around the crosslink (‘unhooking’), possibly associated by single‐stranded DNA degradation. Checkpoint proteins that respond to single‐stranded DNA and double strand breaks participate as important signalling factors that also may attract repair proteins. Many known proteins involved are listed but details remain unknown.

Figure 4.

Model for replication‐coupled DNA crosslink repair, based on studies of a cell‐free system. (a) Plasmid substrate, containing a single ICL in a defined position. (b) Electron microscopy analysis of replication of this substrate, following cell extract addition. (c) Scheme for crosslink processing (see text for details). Note that in contrast to Figure , the assumption of converging and stalled replication forks leads to simultaneous double‐strand breakage and unhooking of the crosslink. See Figure for a similar scheme that includes candidate proteins. Reproduced with permission from Räschle et al. (). © Cell Press and Elsevier.

Figure 5.

Model for replication‐coupled DNA crosslink repair, incorporating studies on Fanconi syndrome proteins. Following stabilisation of the stalled replication fork, the FA core complex results in monoubiquitination of FANCI and FANCD2. Through binding of the UBZ4 domain‐containing protein, SLX4 (= FANCP), ERCC1XPF, MUS81‐EME1 or UBZ4 containing FAN1 may cut in sequential or redundant fashion to unhook the crosslink. TLS is initiated, followed by HR. FANCD1, J, N, O represent recombination proteins. The unhooked crosslink may undergo complete removal by NER. Reproduced from Kim and D'Andrea (). © Creative Commons.



Adamo A, Collis SJ, Adelman CA et al. (2010) Preventing nonhomologous end joining suppresses DNA repair defects of Fanconi anemia. Molecular Cell 39: 25–35.

Akkari YMN, Bateman RL, Reifsteck CA, Olson SB and Grompe M (2000) DNA replication is required to elicit cellular responses to psoralen‐induced DNA interstrand cross‐links. Molecular and Cellular Biology 20: 8283–8289.

Andreassen PR, D'Andrea AD and Taniguchi T (2004) ATR couples FANCD2 monoubiquitination to the DNA‐damage response. Genes and Development 18: 1958–1963.

Bae JB, Mukhopadhyay SS, Liu L et al. (2008) Snm1B/Apollo mediates replication fork collapse and S phase checkpoint activation in response to DNA interstrand cross‐links. Oncogene 27: 5045–5056.

Berardini M, Foster PL and Loechler EL (1999) DNA polymerase II (polB) is involved in a new DNA repair pathway for DNA interstrand cross‐links in E. coli. Journal of Bacteriology 181: 2878–2882.

Bessho T, Mu D and Sancar A (1997) Initiation of DNA interstrand cross‐link repair in humans: the nucleotide excision repair system makes dual incisions 5′ to the cross‐linked base and removes a 22‐ to 28‐nucleotide‐long damage‐free strand. Molecular and Cellular Biology 17: 6822–6830.

Chu G (1994) Cellular responses to cisplatin. Journal of Biological Chemistry 269: 787–790.

Cybulski KE and Howlett NG (2011) FANCP/SLX4: a Swiss army knife of DNA interstrand crosslink repair. Cell Cycle 10: 1757–1763.

Deans AJ and West SC (2011) DNA interstrand crosslink repair and cancer. Nature Reviews Cancer 11: 467–480.

De Silva IU, McHugh PJ, Clingen PH and Hartley JA (2000) Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross‐links in mammalian cells. Molecular and Cellular Biology 20: 7980–7990.

Dronkert ML and Kanaar R (2001) Repair of DNA interstrand cross‐links. Mutation Research 486: 217–247.

Friedberg EC, Walker GC, Siede W et al. (2006) DNA Repair and Mutagenesis, 2nd edn. Washington, DC: American Society of Microbiology Press.

Grossmann KF, Ward AM, Matkovic ME, Folias AE and Moses RE (2001) S. cerevisiae has three pathways for DNA interstrand crosslink repair. Mutation Research 487: 73–83.

Hanada K, Budzowska M, Modesti M et al. (2006) The structure‐specific endonuclease Mus81‐Eme1 promotes conversion of interstrand DNA crosslinks into double‐strands breaks. EMBO Journal 25: 4921–4932.

van Houten B, Gamper H, Holbrook SR, Hearst JE and Sancar A (1986) Action mechanism of ABC excision nuclease on a DNA substrate containing a psoralen crosslink at a defined position. Proceedings of the National Academy of Sciences of the USA 83: 8077–8081.

Hwang IG, Ahn MJ, Park BB et al. (2008) ERCC1 expression as a prognostic marker in N2(+) nonsmall‐cell lung cancer patients treated with platinum‐based neoadjuvant concurrent chemoradiotherapy. Cancer 113: 1379–1386.

Jachymczyk WJ, von Borstel RC, Mowat MR and Hastings PJ (1981) Repair of interstrand cross‐links in DNA of Saccharomyces cerevisiae requires two systems for DNA repair: the RAD3 system and the RAD51 system. Molecular and General Genetics 182: 196–205.

Kim H and D'Andrea AD (2012) Regulation of DNA cross‐link repair by the Fanconi anemia/BRCA pathway. Genes & Development 26: 1393–1408.

Kim H, Yang K, Dejsuphong D and D'Andrea AD (2012) Regulation of Rev1 by the Fanconi anemia core complex. Nature Structural and Molecular Biology 19: 164–170.

Knipscheer P, Räschle M, Smogorzewska A et al. (2009) The Fanconi anemia pathway promotes replication‐dependent DNA interstrand cross‐link repair. Science 326: 1698–1701.

Kumaresan KR, Hang B and Lambert MW (1995) Human endonucleolytic incision of DNA 3′ and 5′ to a site‐directed psoralen monoadduct and interstrand cross‐link. Journal of Biological Chemistry 270: 30709–30716.

Langevin F, Crossan GP, Rosado IV, Arends MJ and Patel KJ (2011) Fancd2 counteracts the toxic effects of naturally produced aldehydes in mice. Nature 475: 53–58.

Lehoczky P, McHugh PJ and Chovanec M (2007) DNA interstrand cross‐link repair in Saccharomyces cerevisiae. FEMS Microbiological Reviews 31: 109–133.

Li X, Hejna J and Moses R (2005) The yeast Snm1 protein is a DNA 5′‐exonuclease. DNA Repair (Amsterdam) 4: 163–170.

MacKay C, Déclais AC, Lundin C et al. (2010) Identification of KIAA1018/FAN1, a DNA repair nuclease recruited to DNA damage by monoubiquitinated FANCD2. Cell 142: 65–76.

Magaña‐Schwencke N, Henriques JAP, Chanet R and Moustacchi E (1982) The fate of 8‐methoxypsoralen photoinduced cross‐links in nuclear and mitochondrial yeast DNA: comparison of wild type and repair deficient strain. Proceedings of the National Academy of Sciences of the USA 79: 1722–1726.

Mamenta EL, Poma EE, Kaufmann WK et al. (1994) Enhanced replicative bypass of platinum‐DNA adducts in cisplatin‐resistant human ovarian carcinoma cell lines. Cancer Research 54: 3500–3505.

Moldovan GL, Madhavan MV, Mirchandani KD et al. (2010) DNA polymerase POLN participates in cross‐link repair and homologous recombination. Molecular and Cellular Biology 30: 1088–1096.

Mu D, Bessho T, Nechev LV et al. (2000) DNA interstrand cross‐links induce futile repair synthesis in mammalian cell extracts. Molecular and Cellular Biology 20: 2446–2454.

Nakanishi K, Yang YG, Pierce AJ et al. (2005) Human Fanconi anemia monoubiquitination pathway promotes homologous DNA repair. Proceedings of the National Academy of Sciences of the USA 102: 1110–1115.

Niedernhofer LJ, Odijk H, Budzowska M et al. (2004) The structure‐specific endonuclease Ercc1‐Xpf is required to resolve DNA interstrand cross‐link‐induced double‐strand breaks. Molecular and Cellular Biology 24: 5776–5787.

Noll DM, Noronha AM, Wilds CJ and Miller PS (2004) Preparation of interstrand cross‐linked DNA oligonucleotide duplexes. Frontiers in Bioscience 9: 421–437.

Oestergaard VH, Langevin F, Kuiken HJ et al. (2007) Deubiquitination of FANCD2 is required for DNA crosslink repair. Molecular Cell 28: 798–809.

Pichierri P and Rosselli F (2004) The DNA crosslink‐induced S‐phase checkpoint depends on ATR‐CHK1 and ATR‐NBS1‐FANCD2 pathways. EMBO Journal 23: 1178–1187.

Räschle M, Knipscheer P, Enoiu M et al. (2008) Mechanism of replication‐coupled DNA interstrand crosslink repair. Cell 134: 969–980.

Rink S, Solomon M, Taylor M et al. (1993) Covalent structure of nitrogen mustard‐induced DNA interstrand crosslink: an N7‐to‐N7 linkage of deoxyguanosine residues at the duplex sequence 5′‐d(GNC). Journal of the American Chemical Society 115: 2551–2557.

Ruhland A, Kircher M, Wilborn F and Brendel M (1981) A yeast mutant specifically sensitive to bifunctional alkylation. Mutation Research 91: 457–462.

Sarkar S, Davies AA, Ulrich HD and McHugh PJ (2006) DNA interstrand crosslink repair during G1 involves nucleotide excision repair and DNA polymerase zeta. EMBO Journal 25: 1285–1294.

Shachar S, Ziv O, Avkin S et al. (2009) Two‐polymerase mechanisms dictate error‐free and error‐prone translesion DNA synthesis in mammals. EMBO Journal 28: 383–393.

Shen X, Jun S, O'Neal LE et al. (2006) REV3 and REV1 play major roles in recombination‐independent repair of DNA interstrand cross‐links mediated by monoubiquitinated proliferating cell nuclear antigen (PCNA). Journal of Biological Chemistry 281: 13869–13872.

Sladek FM, Munn MM, Rupp WD and Howard‐Flanders P (1989) In vitro repair of psoralen‐DNA cross‐links by RecA, UvrABC, and the 5′‐exonuclease of DNA polymerase I. Journal of Biological Chemistry 264: 6755–6765.

Smeaton MB, Hlavin EM, McGregor Mason T et al. (2008) Distortion‐dependent unhooking of interstrand cross‐links in mammalian cell extracts. Biochemistry 47: 9920–9930.

Takata M, Sasaki MS, Tachiri S et al. (2001) Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs. Molecular and Cellular Biology 21: 2858–2866.

Vos J‐MH and Hanawalt PC (1987) Processing of psoralen adducts in an active human gene: repair and replication of DNA containing monoadducts and interstrand cross‐links. Cell 50: 789–799.

Wu F, Lin X, Okuda T and Howell SB (2004) DNA polymerase zeta regulates cisplatin cytotoxicity, mutagenicity, and the rate of development of cisplatin resistance. Cancer Research 64: 8029–8035.

Wu Q, Christensen LA, Legerski RJ and Vasquez KM (2005) Mismatch repair participates in error‐free processing of DNA interstrand crosslinks in human cells. EMBO Reports 6: 551–557.

Wu Q and Vasquez KM (2008) Human MLH1 protein participates in genomic damage checkpoint signaling in response to DNA interstrand crosslinks, while MSH2 functions in DNA repair. PLoS Genetics 4: e1000189.

Zhang N, Kaur R, Lu X et al. (2005) The Pso4 mRNA splicing and DNA repair complex interacts with WRN for processing of DNA interstrand cross‐links. Journal of Biological Chemistry 280: 40559–40567.

Zhang N, Liu X, Li L and Legerski R (2007) Double‐strand breaks induce homologous recombinational repair of interstrand cross‐links via cooperation of MSH2, ERCC1‐XPF, REV3, and the Fanconi anemia pathway. DNA Repair (Amsterdam) 6: 1670–1678.

Zhang N, Lu X, Zhang X, Peterson CA and Legerski RJ (2002) hMutSbeta is required for the recognition and uncoupling of psoralen interstrand cross‐links in vitro. Molecular and Cellular Biology 22: 2388–2397.

Zheng H, Wang X, Warren AJ et al. (2003) Nucleotide excision repair‐ and polymerase eta‐mediated error‐prone removal of mitomycin C interstrand cross‐links. Molecular and Cellular Biology 23: 754–761.

Further Reading

Basu A and Krishnamurthy S (2010) Cellular responses to cisplatin‐induced DNA damage. Journal of Nucleic Acids 2010: 1–16.

Beljanski V, Marzilli LG and Doetsch PW (2004) DNA damage‐processing pathways involved in the eukaryotic cellular response to anticancer DNA cross‐linking drugs. Molecular Pharmacology 65: 1496–1506.

Brendel M and Ruhland A (1984) Relationships between functionality and genetic toxicology of selected DNA damaging agents. Mutation Research 133: 51–85.

Kohn KW (1996) Beyond DNA cross‐linking: history and prospects of DNA targeted cancer treatment. Cancer Research 56: 5533–5546.

Lange SS, Takata K and Wood RD (2011) DNA polymerases and cancer. Nature Reviews Cancer 11: 96–110.

Roberts JJ and Thomson AJ (1979) The mechanism of action of antitumour platinum compounds. Progress in Nucleic Acid Research and Molecular Biology 22: 71–133.

Siddik ZH (2003) Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 22: 7265–7279.

Song PS and Tapley KJ (1979) Photochemistry and photobiology of psoralens. Photochemistry and Photobiology 29: 1177–1197.

Wang W (2007) Emergence of a DNA‐damage response network consisting of Fanconi anaemia and BRCA proteins. Nature Reviews Genetics 8: 735–748.

Wood RD (2010) Mammalian nucleotide excision repair proteins and interstrand crosslink repair. Environmental and Molecular Mutagenesis 51: 520–526.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

How to Cite close
Siede, Wolfram(Aug 2014) DNA Interstrand Crosslink Repair. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0000575.pub3]