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Molecular and Cellular Biology, February 2001, p. 713-720, Vol. 21, No. 3
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.3.713-720.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Involvement of Nucleotide Excision Repair in a Recombination-Independent and Error-Prone Pathway of DNA Interstrand Cross-Link Repair

Xin Wang,1 Carolyn A. Peterson,2 Huyong Zheng,1 Rodney S. Nairn,3 Randy J. Legerski,2 and Lei Li1,*

Departments of Experimental Radiation Oncology,1 Molecular Genetics,2 and Carcinogenesis,3 The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Received 24 July 2000/Returned for modification 24 August 2000/Accepted 26 October 2000

DNA interstrand cross-links (ICLs) block the strand separation necessary for essential DNA functions such as transcription and replication and, hence, represent an important class of DNA lesion. Since both strands of the double helix are affected in cross-linked DNA, it is likely that conservative recombination using undamaged homologous regions as a donor may be required to repair ICLs in an error-free manner. However, in Escherichia coli and yeast, recombination-independent mechanisms of ICL repair have been identified in addition to recombinational repair pathways. To study the repair mechanisms of interstrand cross-links in mammalian cells, we developed an in vivo reactivation assay to examine the removal of interstrand cross-links in cultured cells. A site-specific psoralen cross-link was placed between the promoter and the coding region to inactivate the expression of green fluorescent protein or luciferase genes from reporter plasmids. By monitoring the reactivation of the reporter gene, we showed that a single defined psoralen cross-link was removed in repair-proficient cells in the absence of undamaged homologous sequences, suggesting the existence of an ICL repair pathway that is independent of homologous recombination. Mutant cell lines deficient in the nucleotide excision repair pathway were examined and found to be highly defective in the recombination-independent repair of ICLs, while mutants deficient in homologous recombination were found to be proficient. Mutation analysis of plasmids recovered from transfected cells showed frequent base substitutions at or near positions opposing a cross-linked thymidine residue. Based on these results, we suggest a distinct pathway for DNA interstrand cross-link repair involving nucleotide excision repair and a putative lesion bypass mechanism.

* Corresponding author. Mailing address: The University of Texas M. D. Anderson Cancer Center, Box 66, 1515 Holcombe Blvd., Houston, TX 77030. Phone: (713) 792-3424. Fax: (716) 794-5369. E-mail: leili{at}mdanderson.org.

Molecular and Cellular Biology, February 2001, p. 713-720, Vol. 21, No. 3
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.3.713-720.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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