scholarly journals Repair of Intermediate Structures Produced at DNA Interstrand Cross-Links in Saccharomyces cerevisiae

2000 ◽  
Vol 20 (10) ◽  
pp. 3425-3433 ◽  
Author(s):  
Peter J. McHugh ◽  
William R. Sones ◽  
John A. Hartley
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Antitumor Agents ◽  
Excision Repair ◽  
Alkylating Agents ◽  
Exponential Growth Phase ◽  
Cellular Mechanisms ◽  
Nucleotide Excision ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Cycle Dependent

ABSTRACT Bifunctional alkylating agents and other drugs which produce DNA interstrand cross-links (ICLs) are among the most effective antitumor agents in clinical use. In contrast to agents which produce bulky adducts on only one strand of the DNA, the cellular mechanisms which act to eliminate DNA ICLs are still poorly understood, although nucleotide excision repair is known to play a crucial role in an early repair step. Using haploid Saccharomyces cerevisiae strains disrupted for genes central to the recombination, nonhomologous end-joining (NHEJ), and mutagenesis pathways, all these activities were found to be involved in the repair of nitrogen mustard (mechlorethamine)- and cisplatin-induced DNA ICLs, but the particular pathway employed is cell cycle dependent. Examination of whole chromosomes from treated cells using contour-clamped homogenous electric field electrophoresis revealed the intermediate in the repair of ICLs in dividing cells, which are mostly in S phase, to be double-strand breaks (DSBs). The origin of these breaks is not clear since they were still efficiently induced in nucleotide excision and base excision repair-deficient, mismatch repair-defective,rad27 and mre11 disruptant strains. In replicating cells, RAD52-dependent recombination and NHEJ both act to repair the DSBs. In contrast, few DSBs were observed in quiescent cells, and recombination therefore seems dispensable for repair. The activity of the Rev3 protein (DNA polymerase ζ) is apparently more important for the processing of intermediates in stationary-phase cells, since rev3 disruptants were more sensitive in this phase than in the exponential growth phase.

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.

scholarly journals Cho Endonuclease Functions during DNA Interstrand Cross-Link Repair in Escherichia coli

2016 ◽  
Vol 198 (22) ◽  
pp. 3099-3108 ◽  
Author(s):  
Anthonige Vidya Perera ◽  
James Brian Mendenhall ◽  
Charmain Tan Courcelle ◽  
Justin Courcelle
Keyword(s):  
Nucleotide Excision Repair ◽  
Excision Repair ◽  
Repair Process ◽  
Differential Sensitivity ◽  
Duplex Dna ◽  
Dna Lesion ◽  
Nucleotide Excision ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Complex Lesions

ABSTRACTDNA interstrand cross-links are complex lesions that covalently link both strands of the duplex DNA. Lesion removal is proposed to be initiated via the UvrABC nucleotide excision repair complex; however, less is known about the subsequent steps of this complex repair pathway. In this study, we characterized the contribution of nucleotide excision repair mutants to survival in the presence of psoralen-induced damage. Unexpectedly, we observed that the nucleotide excision repair mutants exhibit differential sensitivity to psoralen-induced damage, withuvrCmutants being less sensitive than eitheruvrAoruvrB. We show that Cho, an alternative endonuclease, acts with UvrAB and is responsible for the reduced hypersensitivity ofuvrCmutants. We find that Cho's contribution to survival correlates with the presence of DNA interstrand cross-links, rather than monoadducts, and operates at a step after, or independently from, the initial incision during the global repair of psoralen DNA adducts from the genome.IMPORTANCEDNA interstrand cross-links are complex lesions that covalently bind to both strands of the duplex DNA and whose mechanism of repair remains poorly understood. In this study, we show that Cho, an alternative endonuclease, acts with UvrAB and participates in the repair of DNA interstrand cross-links formed in the presence of photoactivated psoralens. Cho's contribution to survival correlates with the presence of DNA interstrand cross-links and operates at a step after, or independently from, the initial incision during the repair process.

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.

scholarly journals Nucleotide Excision Repair- and Polymerase η-Mediated Error-Prone Removal of Mitomycin C Interstrand Cross-Links

2003 ◽  
Vol 23 (2) ◽  
pp. 754-761 ◽  
Author(s):  
Huyong Zheng ◽  
Xin Wang ◽  
Amy J. Warren ◽  
Randy J. Legerski ◽  
Rodney S. Nairn ◽  
...  
Keyword(s):  
Mitomycin C ◽  
Nucleotide Excision Repair ◽  
Mammalian Cells ◽  
Excision Repair ◽  
Double Helix ◽  
Dna Lesions ◽  
Nucleotide Excision ◽  
Interstrand Cross Links ◽  
Cross Links

ABSTRACT Interstrand cross-links (ICLs) make up a unique class of DNA lesions in which both strands of the double helix are covalently joined, precluding strand opening during replication and transcription. The repair of DNA ICLs has become a focus of study since ICLs are recognized as the main cytotoxic lesion inflicted by an array of alkylating compounds used in cancer treatment. As is the case for double-strand breaks, a damage-free homologous copy is essential for the removal of ICLs in an error-free manner. However, recombination-independent mechanisms may exist to remove ICLs in an error-prone fashion. We have developed an in vivo reactivation assay that can be used to examine the removal of site-specific mitomycin C-mediated ICLs in mammalian cells. We found that the removal of the ICL from the reporter substrate could take place in the absence of undamaged homologous sequences in repair-proficient cells, suggesting a cross-link repair mechanism that is independent of homologous recombination. Systematic analysis of nucleotide excision repair mutants demonstrated the involvement of transcription-coupled nucleotide excision repair and a partial requirement for the lesion bypass DNA polymerase η encoded by the human POLH gene. From these observations, we propose the existence of a recombination-independent and mutagenic repair pathway for the removal of ICLs in mammalian cells.

scholarly journals Characterization of the Mycobacterial NER System Reveals Novel Functions of the uvrD1 Helicase

2008 ◽  
Vol 191 (2) ◽  
pp. 555-562 ◽  
Author(s):  
Carolin Güthlein ◽  
Roger M. Wanner ◽  
Peter Sander ◽  
Elaine O. Davis ◽  
Martin Bosshard ◽  
...  
Keyword(s):  
Excision Repair ◽  
Repair System ◽  
Double Knockout ◽  
Dna Damaging Agents ◽  
Nucleotide Excision ◽  
Mismatch Repair System ◽  
Interstrand Cross Links ◽  
Cross Links ◽  
Bulky Dna Adducts

ABSTRACT In this study, we investigated the role of the nucleotide excision repair (NER) pathway in mycobacterial DNA repair. Mycobacterium smegmatis lacking the NER excinuclease component uvrB or the helicase uvrD1 gene and a double knockout lacking both genes were constructed, and their sensitivities to a series of DNA-damaging agents were analyzed. As anticipated, the mycobacterial NER system was shown to be involved in the processing of bulky DNA adducts and interstrand cross-links. In addition, it could be shown to exert a protective effect against oxidizing and nitrosating agents. Interestingly, inactivation of uvrB and uvrD1 significantly increased marker integration frequencies in gene conversion assays. This implies that in mycobacteria (which lack the postreplicative mismatch repair system) NER, and particularly the UvrD1 helicase, is involved in the processing of a subset of recombination-associated mismatches.

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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