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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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