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Molecular and Cellular Biology, August 1999, p. 5619-5630, Vol. 19, No. 8
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Interstrand Cross-Links Induce DNA Synthesis in Damaged and Undamaged Plasmids in Mammalian Cell Extracts

Lei Li,1 Carolyn A. Peterson,2 Xiaoyan Lu,2 Ping Wei,2,dagger and Randy J. Legerski2,*

Departments of Experimental Radiation Oncology1 and Molecular Genetics,2 University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

Received 19 February 1999/Returned for modification 25 March 1999/Accepted 19 May 1999

Mammalian cell extracts have been shown to carry out damage-specific DNA repair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-link induces DNA synthesis in both the damaged plasmid and a second homologous unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly stimulates repair synthesis in the cross-linked plasmid. Heterologous DNAs also stimulate repair synthesis to variable extents. Psoralen monoadducts and double-strand breaks do not induce repair synthesis in the unmodified plasmid, indicating that such incorporation is specific to interstrand cross-links. This induced repair synthesis is consistent with previous evidence indicating a recombinational mode of repair for interstrand cross-links. DNA synthesis is compromised in extracts from mutants (deficient in ERCC1, XPF, XRCC2, and XRCC3) which are all sensitive to DNA cross-linking agents but is normal in extracts from mutants (XP-A, XP-C, and XP-G) which are much less sensitive. Extracts from Fanconi anemia cells exhibit an intermediate to wild-type level of activity dependent upon the complementation group. The DNA synthesis deficit in ERCC1- and XPF-deficient extracts is restored by addition of purified ERCC1-XPF heterodimer. This system provides a biochemical assay for investigating mechanisms of interstrand cross-link repair and should also facilitate the identification and functional characterization of cellular proteins involved in repair of these lesions.

* Corresponding author. Mailing address: Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030. Phone: (713) 792-8941. Fax: (713) 794-4295. E-mail: randy_legerski{at}molgen.mdacc.tmc.edu.

dagger Present Address: Department of Cell Biology, Baylor College of Medicine, Houston, TX 77030.

Molecular and Cellular Biology, August 1999, p. 5619-5630, Vol. 19, No. 8
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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