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 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 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.

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.

FANCD2 Localizes to Cross-Linked Induced Nuclear Foci That Are Distinct From Those to Which αaII Spectrin and the Cross-Link Repair Protein, XPF, Co-Localize, Indicating An Involvement of These Proteins in Different Steps of the DNA Interstrand Cross-Link Repair Process.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3196-3196
Author(s):  
Pan Zhang ◽  
Deepa Sridharan ◽  
Michael Acosta ◽  
Muriel Lambert
Keyword(s):  
Time Course ◽  
Repair Process ◽  
Myelogenous Leukemia ◽  
Cross Linking ◽  
Cross Link ◽  
Repair Protein ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Nuclear Foci ◽  
The Cross

Abstract Abstract 3196 Poster Board III-133 The hereditary bone marrow failure disorder, Fanconi anemia (FA), is characterized by a markedly increased incidence of acute myelogenous leukemia, diverse congenital abnormalities and a defect in ability to repair DNA interstrand cross-links. We have previously shown that in FA cells there is a deficiency in the structural protein nonerythroid a spectrin (aSpII), which is involved in repair of DNA interstrand cross-links and binds to cross-linked DNA. aSpII co-localizes in nuclear foci with FANCA and the cross-link repair protein, XPF, after normal human cells are damaged with a DNA interstrand cross-linking agent. One of the FA proteins which is thought to play an important role in the repair of DNA interstrand cross-links is FANCD2, which is known to form nuclear foci after cross-link damage. The present study was undertaken in order to get a better understanding of the relationship between aSpII and FANCD2, whether they interact with each other during the DNA repair process and co-localize in damage-induced nuclear foci. Immunofluorescence microscopy was carried out to determine whether these proteins co-localized in nuclear foci after cells were damaged with a DNA interstrand cross-linking agent, 8-methylpsoralen plus UVA light (8-MOP) or mitomycin C (MMC). Time course measurements showed that FANCD2 foci were first visible at 2 hours after damage and increased up to 16 hours and were still present at 72 hours after damage. This time course of foci formation correlated with levels of monoubiquitination of FANCD2. Measurement of gH2AX foci formation showed that the time course of foci formation was similar to that of FANCD2 measured up to 72 hours post damage. In contrast, aSpII foci were first visible between 8-10 hours after damage. The number of these foci peaked at 16 hours and by 24 hours foci were no longer observed. Co-localization studies showed that there was little co-localization of the FANCD2 and aSpII foci over this time course. This indicates that these two proteins may be involved in different steps in the DNA interstrand cross-link repair process. Based on models that have been proposed for the role of FANCD2 in the repair of DNA interstrand cross-links, we propose that, after DNA damage, FANCD2 localizes at DNA replication forks stalled at sites of interstrand cross-links and aids in the assembly of proteins at this site. This is followed by localization of aSpII and XPF and other proteins involved in the initial incision steps in DNA interstrand cross-link repair where they play a role in the unhooking of the cross-link. FANCD2 is then involved in subsequent steps in the repair process, which involve homologous recombination. Thus two proteins, FANCD2 and aSpII, both of which have been shown to be critical for the DNA interstrand cross-link repair process may be involved in different or distinct steps in this repair process. Deficiencies in these proteins would impact on DNA interstrand cross-link repair and, as we have shown for aIISp, would have an adverse effect on the genomic stability of FA cells. . Disclosures No relevant conflicts of interest to declare.

scholarly journals Processing of a Psoralen DNA Interstrand Cross-link by XPF-ERCC1 Complex in Vitro

2007 ◽  
Vol 283 (3) ◽  
pp. 1275-1281 ◽  
Author(s):  
Laura A. Fisher ◽  
Mika Bessho ◽  
Tadayoshi Bessho
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Dependent Manner ◽  
Cross Link ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
The Cross ◽  
Stalled Fork

The processing of stalled forks caused by DNA interstrand cross-links (ICLs) has been proposed to be an important step in initiating mammalian ICL repair. To investigate a role of the XPF-ERCC1 complex in this process, we designed a model substrate DNA with a single psoralen ICL at a three-way junction (Y-shaped DNA), which mimics a stalled fork structure. We found that the XPF-ERCC1 complex makes an incision 5′ to a psoralen lesion on Y-shaped DNA in a damage-dependent manner. Furthermore, the XPF-ERCC1 complex generates an ICL-specific incision on the 3′-side of an ICL. The ICL-specific 3′-incision, along with the 5′-incision, on the cross-linked Y-shaped DNA resulted in the separation of the two cross-linked strands (the unhooking of the ICL) and the induction of a double strand break near the cross-linked site. These results implicate the XPF-ERCC1 complex in initiating ICL repair by unhooking the ICL, which simultaneously induces a double strand break at a stalled fork.

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.

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

2001 ◽  
Vol 21 (3) ◽  
pp. 713-720 ◽  
Author(s):  
Xin Wang ◽  
Carolyn A. Peterson ◽  
Huyong Zheng ◽  
Rodney S. Nairn ◽  
Randy J. Legerski ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Nucleotide Excision Repair ◽  
Mammalian Cells ◽  
Cultured Cells ◽  
Excision Repair ◽  
Repair Pathway ◽  
Cross Link ◽  
Nucleotide Excision ◽  
Interstrand Cross Links ◽  
Cross Links

ABSTRACT 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.

Fast, inexpensive, high-yielding, site-selective, chemical synthesis of cross-linked DNA duplexes via hydrazone formation between N[superscript 4]-aminocytidine and Ap-sites

2017 ◽  
Author(s):  
◽  
Jacqueline Gamboa Varela
Keyword(s):  
High Yield ◽  
Dna Duplexes ◽  
Cross Link ◽  
University Of Missouri ◽  
Cellular Processes ◽  
Abasic Sites ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
The Cross ◽  
High Yields

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] DNA is the central molecule of biology as it stores the genetic information for cells to properly function and develop. Modifications to the DNA can stall cellular processes such as replication and transcription, leading the cell to recruit repair machinery or in some cases undergo apoptosis. Interstrand cross-links are particularly significant types of DNA damage because they prevent strand separation required for replication and transcription. Cross-links involve bonding between the two strands of DNA. The rate and mechanism of cross-link repair in cells are not well understood. A significant challenge in the study of cross-link repair is the synthesis of chemically well-defined DNA cross-links. Here we summarize the preparation of cross-links derived from the hydrazone formation between a non-natural nucleobase N4-aminocytidine and abasic sites in duplex DNA. The cross-link was generated rapidly and in high yield. The cross-link is stable under physiological conditions but, interestingly, can be reversibly dissociated and re-formed by thermal cycling between 20-80 [degrees]C. We provided evidence that the cross-link is stable against multiple agents and the cross-link is reversible. We used this chemistry to prepare structurally diverse cross-links for the utilization in cross-link repair studies. Overall, we developed a synthetic cross-link that is easily and rapidly prepared from commercially available reagents in high yields, at defined locations in duplexed DNA.

scholarly journals DNA Polymerase II (polB) Is Involved in a New DNA Repair Pathway for DNA Interstrand Cross-Links inEscherichia coli

1999 ◽  
Vol 181 (9) ◽  
pp. 2878-2882 ◽  
Author(s):  
Mark Berardini ◽  
Patricia L. Foster ◽  
Edward L. Loechler
Keyword(s):  
Dna Polymerase ◽  
Nitrogen Mustard ◽  
Wild Type ◽  
Cross Link ◽  
Replication Efficiency ◽  
E Coli ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
The Cross ◽  
Dna Polymerase Ii

ABSTRACT DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escherichia coli, and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be ∼0.6. Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination. (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in apolB + (∼0.6) than in a ΔpolB(∼0.1) strain. Complementation with apolB +-containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate experiments, E. coli was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type <polB < recA < polB recA ∼ uvrA. Because cells deficient in either recombination (recA) or DNA polymerase II (polB) are hypersensitive to nitrogen mustard killing,E. coli appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway. Furthermore, these results show that some cross-links are uniquely repaired by each pathway. This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in E. coli. It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.

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.

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