Generation of single-nucleotide repair patches following excision of uracil residues from DNA.

The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase beta.

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Detection and Measurement of Nucleotide Excision Repair Synthesis by Mammalian Cell Extracts in Vitro

Methods ◽

10.1006/meth.1995.1022 ◽

1995 ◽

Vol 7 (2) ◽

pp. 163-175 ◽

Cited By ~ 51

Author(s):

Richard D. Wood ◽

Maureen Biggerstaff ◽

Mahmud K.K. Shivji

Keyword(s):

Nucleotide Excision Repair ◽

Mammalian Cell ◽

Excision Repair ◽

Repair Synthesis ◽

Cell Extracts ◽

Nucleotide Excision

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Mutations in XPA that prevent association with ERCC1 are defective in nucleotide excision repair.

Molecular and Cellular Biology ◽

10.1128/mcb.15.4.1993 ◽

1995 ◽

Vol 15 (4) ◽

pp. 1993-1998 ◽

Cited By ~ 86

Author(s):

L Li ◽

C A Peterson ◽

X Lu ◽

R J Legerski

Keyword(s):

Nucleotide Excision Repair ◽

Excision Repair ◽

Repair Synthesis ◽

Damage Site ◽

Cell Extracts ◽

Nucleotide Excision ◽

Delta G ◽

Dna Repair Synthesis

The human repair proteins XPA and ERCC1 have been shown to be absolutely required for the incision step of nucleotide excision repair, and recently we identified an interaction between these two proteins both in vivo and in vitro (L. Li, S. J. Elledge, C. A. Peterson, E. S. Bales, and R. J. Legerski, Proc. Natl. Acad. Sci. USA 91:5012-5016, 1994). In this report, we demonstrate the functional relevance of this interaction. The ERCC1-binding domain on XPA was previously mapped to a region containing two highly conserved XPA sequences, Gly-72 to Phe-75 and Glu-78 to Glu-84, which are termed the G and E motifs, respectively. Site-specific mutagenesis was used to independently delete these motifs and create two XPA mutants referred to as delta G and delta E. In vitro, the binding of ERCC1 to delta E was reduced by approximately 70%, and binding to delta G was undetectable; furthermore, both mutants failed to complement XPA cell extracts in an in vitro DNA repair synthesis assay. In vivo, the delta E mutant exhibited an intermediate level of complementation of XPA cells and the delta G mutant exhibited little or no complementation. In addition, the delta G mutant inhibited repair synthesis in wild-type cell extracts, indicating that it is a dominant negative mutant. The delta E and delta G mutations, however, did not affect preferential binding of XPA to damaged DNA. These results suggest that the association between XPA and ERCC1 is a required step in the nucleotide excision repair pathway and that the probable role of the interaction is to recruit the ERCC1 incision complex to the damage site. Finally, the affinity of the XPA-ERCC1 complex was found to increase as a function of salt concentration, indicating a hydrophobic interaction; the half-life of the complex was determined to be approximately 90 min.

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In Vitro Base Excision Repair Assay Using Mammalian Cell Extracts

DNA Repair Protocols ◽

10.1007/978-1-4612-1608-7_24 ◽

1999 ◽

pp. 301-315 ◽

Cited By ~ 5

Author(s):

Guido Frosina ◽

Enrico Cappelli ◽

Paola Fortini ◽

Eugenia Dogliotti

Keyword(s):

Mammalian Cell ◽

Base Excision Repair ◽

Excision Repair ◽

Cell Extracts ◽

Base Excision

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In Vitro Base Excision Repair Assay Using Mammalian Cell Extracts

DNA Repair Protocols ◽

10.1385/1-59259-675-4:301 ◽

2003 ◽

pp. 301-315 ◽

Cited By ~ 2

Author(s):

Guido Frosina ◽

Enrico Cappelli ◽

Paola Fortini ◽

Eugenia Dogliotti

Keyword(s):

Mammalian Cell ◽

Base Excision Repair ◽

Excision Repair ◽

Cell Extracts ◽

Base Excision

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

Molecular and Cellular Biology ◽

10.1128/mcb.19.8.5619 ◽

1999 ◽

Vol 19 (8) ◽

pp. 5619-5630 ◽

Cited By ~ 42

Author(s):

Lei Li ◽

Carolyn A. Peterson ◽

Xiaoyan Lu ◽

Ping Wei ◽

Randy J. Legerski

Keyword(s):

Dna Synthesis ◽

Mammalian Cell ◽

Functional Characterization ◽

Complementation Group ◽

Cross Link ◽

Repair Synthesis ◽

Cell Extracts ◽

Interstrand Cross Links ◽

Cross Links ◽

Dna Repair Synthesis

ABSTRACT 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.

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DNA Synthesis and dRPase Activities of Polymerase β Are Both Essential for Single-Nucleotide Patch Base Excision Repair in Mammalian Cell Extracts

Biochemistry ◽

10.1021/bi002064s ◽

2001 ◽

Vol 40 (3) ◽

pp. 809-813 ◽

Cited By ~ 41

Author(s):

Andrej Ja. Podlutsky ◽

Irina I. Dianova ◽

Samuel H. Wilson ◽

Vilhelm A. Bohr ◽

Grigory L. Dianov

Keyword(s):

Dna Synthesis ◽

Mammalian Cell ◽

Base Excision Repair ◽

Excision Repair ◽

Single Nucleotide ◽

Cell Extracts ◽

Base Excision

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hMutSβ Is Required for the Recognition and Uncoupling of Psoralen Interstrand Cross-Links In Vitro

Molecular and Cellular Biology ◽

10.1128/mcb.22.7.2388-2397.2002 ◽

2002 ◽

Vol 22 (7) ◽

pp. 2388-2397 ◽

Cited By ~ 67

Author(s):

Nianxiang Zhang ◽

Xiaoyan Lu ◽

Xiaoshan Zhang ◽

Carolyn A. Peterson ◽

Randy J. Legerski

Keyword(s):

Mismatch Repair ◽

Nucleotide Excision Repair ◽

Mammalian Cell ◽

Mammalian Cells ◽

Excision Repair ◽

Cell Extracts ◽

Nucleotide Excision ◽

Interstrand Cross Links ◽

Cross Links

ABSTRACT The removal of interstrand cross-links (ICLs) from DNA in higher eucaryotes is not well understood. Here, we show that processing of psoralen ICLs in mammalian cell extracts is dependent upon the mismatch repair complex hMutSβ but is not dependent upon the hMutSα complex or hMlh1. The processing of psoralen ICLs is also dependent upon the nucleotide excision repair proteins Ercc1 and Xpf but not upon other components of the excision stage of this pathway or upon Fanconi anemia proteins. Products formed during the in vitro reaction indicated that the ICL has been removed or uncoupled from the cross-linked substrate in the mammalian cell extracts. Finally, the hMutSβ complex is shown to specifically bind to psoralen ICLs, and this binding is stimulated by the addition of PCNA. Thus, a novel pathway for processing ICLs has been identified in mammalian cells which involves components of the mismatch repair and nucleotide excision repair pathways.

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