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

1999 ◽  
Vol 19 (8) ◽  
pp. 5619-5630 ◽  
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.

scholarly journals DNA Interstrand Cross-Links Induce Futile Repair Synthesis in Mammalian Cell Extracts

2000 ◽  
Vol 20 (7) ◽  
pp. 2446-2454 ◽  
Author(s):  
David Mu ◽  
Tadayoshi Bessho ◽  
Lubomir V. Nechev ◽  
David J. Chen ◽  
Thomas M. Harris ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Excision Repair ◽  
Mutant Cell ◽  
Complex Structure ◽  
Cross Link ◽  
Repair Synthesis ◽  
Cell Extracts ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
The Cross

ABSTRACT DNA interstrand cross-links are induced by many carcinogens and anticancer drugs. It was previously shown that mammalian DNA excision repair nuclease makes dual incisions 5′ to the cross-linked base of a psoralen cross-link, generating a gap of 22 to 28 nucleotides adjacent to the cross-link. We wished to find the fates of the gap and the cross-link in this complex structure under conditions conducive to repair synthesis, using cell extracts from wild-type and cross-linker-sensitive mutant cell lines. We found that the extracts from both types of strains filled in the gap but were severely defective in ligating the resulting nick and incapable of removing the cross-link. The net result was a futile damage-induced DNA synthesis which converted a gap into a nick without removing the damage. In addition, in this study, we showed that the structure-specific endonuclease, the XPF-ERCC1 heterodimer, acted as a 3′-to-5′ exonuclease on cross-linked DNA in the presence of RPA. Collectively, these observations shed some light on the cellular processing of DNA cross-links and reveal that cross-links induce a futile DNA synthesis cycle that may constitute a signal for specific cellular responses to cross-linked DNA.

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

1992 ◽  
Vol 12 (4) ◽  
pp. 1605-1612 ◽  
Author(s):  
G Dianov ◽  
A Price ◽  
T Lindahl
Keyword(s):  
Mammalian Cell ◽  
Excision Repair ◽  
Enzymatic Digestion ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Repair Synthesis ◽  
Single Nucleotide ◽  
Cell Extracts ◽  
Dna Repair Synthesis

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.

scholarly journals 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.

1997 ◽  
Vol 17 (12) ◽  
pp. 6822-6830 ◽  
Author(s):  
T Bessho ◽  
D Mu ◽  
A Sancar
Keyword(s):  
Dna Repair ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Repair System ◽  
Cross Link ◽  
Unique Challenge ◽  
Cell Extracts ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
The Cross

Most DNA repair mechanisms rely on the redundant information inherent to the duplex to remove damaged nucleotides and replace them with normal ones, using the complementary strand as a template. Interstrand cross-links pose a unique challenge to the DNA repair machinery because both strands are damaged. To study the repair of interstrand cross-links by mammalian cells, we tested the activities of cell extracts of wild-type or excision repair-defective rodent cell lines and of purified human excision nuclease on a duplex with a site-specific cross-link. We found that in contrast to monoadducts, which are removed by dual incisions bracketing the lesion, the cross-link causes dual incisions, both 5' to the cross-link in one of the two strands. The net result is the generation of a 22- to 28-nucleotide-long gap immediately 5' to the cross-link. This gap may act as a recombinogenic signal to initiate cross-link removal.

scholarly journals Mechanism of DNA interstrand cross-link processing by repair nuclease FAN1

Science ◽  
2014 ◽  
Vol 346 (6213) ◽  
pp. 1127-1130 ◽  
Author(s):  
Renjing Wang ◽  
Nicole S. Persky ◽  
Barney Yoo ◽  
Ouathek Ouerfelli ◽  
Agata Smogorzewska ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Crystal Structures ◽  
Dna Adducts ◽  
Fanconi Anemia ◽  
Biochemical Data ◽  
Degenerative Diseases ◽  
Cross Link ◽  
Interstrand Cross Links ◽  
Cross Links

DNA interstrand cross-links (ICLs) are highly toxic lesions associated with cancer and degenerative diseases. ICLs can be repaired by the Fanconi anemia (FA) pathway and through FA-independent processes involving the FAN1 nuclease. In this work, FAN1-DNA crystal structures and biochemical data reveal that human FAN1 cleaves DNA successively at every third nucleotide. In vitro, this exonuclease mechanism allows FAN1 to excise an ICL from one strand through flanking incisions. DNA access requires a 5′-terminal phosphate anchor at a nick or a 1- or 2-nucleotide flap and is augmented by a 3′ flap, suggesting that FAN1 action is coupled to DNA synthesis or recombination. FAN1’s mechanism of ICL excision is well suited for processing other localized DNA adducts as well.

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

1992 ◽  
Vol 12 (4) ◽  
pp. 1605-1612
Author(s):  
G Dianov ◽  
A Price ◽  
T Lindahl
Keyword(s):  
Mammalian Cell ◽  
Excision Repair ◽  
Enzymatic Digestion ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Repair Synthesis ◽  
Single Nucleotide ◽  
Cell Extracts ◽  
Dna Repair Synthesis

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.

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

2002 ◽  
Vol 22 (7) ◽  
pp. 2388-2397 ◽  
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.

scholarly journals Distortion-Dependent Unhooking of Interstrand Cross-Links in Mammalian Cell Extracts†

Biochemistry ◽  
2008 ◽  
Vol 47 (37) ◽  
pp. 9920-9930 ◽  
Author(s):  
Michael B. Smeaton ◽  
Erica M. Hlavin ◽  
Tracey McGregor Mason ◽  
Anne M. Noronha ◽  
Christopher J. Wilds ◽  
...  
Keyword(s):  
Mammalian Cell ◽  
Cell Extracts ◽  
Interstrand Cross Links ◽  
Cross Links

scholarly journals Localization of DNA repair synthesis by human cell extracts to a short region at the site of a lesion

1989 ◽  
Vol 264 (36) ◽  
pp. 21788-21792
Author(s):  
J Hansson ◽  
M Munn ◽  
W D Rupp ◽  
R Kahn ◽  
R D Wood
Keyword(s):  
Dna Repair ◽  
Human Cell ◽  
Repair Synthesis ◽  
Short Region ◽  
Cell Extracts ◽  
Dna Repair Synthesis

Diverse applications of covalent cross-links in duplex DNA: From anti-cancer drugs to the detection of disease-relevant single nucleotide polymorphisms to the synthesis of well-defined substrates for the study of novel DNA repair processes

2018 ◽  
Author(s):  
◽  
Maryam Imani Nejad
Keyword(s):  
Excision Repair ◽  
Alkylating Agents ◽  
Nucleotide Polymorphisms ◽  
Cross Link ◽  
Single Nucleotide ◽  
Synthetic Dna ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Ap Sites ◽  
Cellular Dna

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Abasic (Ap) sites are a common form of DNA lesion that occur endogenously 50,000-200,000 per cell per day in mammals. The alkylation of the guanine and adenine residues by the alkylating agents such as nitrogen mustards also induces the formation of Ap sites in genomic DNA. Our group recently showed that Ap sites can forge DNA-DNA interstrand cross-links in some sequences via reaction of the Ap aldehyde residue with the exocyclic amino groups of nucleobases, such as adenine and guanine, on the opposing strand of the DNA duplex. The earlier work in the group revealed that formation of these covalent bridges between two DNA strands is highly sequence- dependent. Although interstrand cross-links are one of the most deleterious types of cellular DNA damage, the availability of synthetic DNA duplexes containing chemically well-defined, site-specific interstrand cross-links has been proven to be a valuable tool in biological chemistry and medicine. We prepared and characterized a new Ap-derived interstrand cross-link. In another project, we use these remarkable cross-linking reactions for the covalent capture of disease-relevant single nucleotide polymorphism by using a protein nanopore technology. The complex mechanisms underlying cross-link repair in cells and limited availability of stable and defined cross-link are two major reasons why repair pathways of these lesions are not yet well understood. By preparing a variety of Ap-derived cross-links, we studied the role of a base excision repair DNA glycosylase, NEIL3 in unhooking the lesions.

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