Inhibition of DNA Repair Replication by DNA Binding Drugs Which Sensitize Cells to Alkylating Agents and X-Rays

Exposure of Escherichia coli to alkylating agents activates expression of AidB in addition to DNA repair proteins Ada, AlkA, and AlkB. AidB was recently shown to possess a flavin adenine dinucleotide (FAD) cofactor and to bind to dsDNA, implicating it as a flavin-dependent DNA repair enzyme. However, the molecular mechanism by which AidB acts to reduce the mutagenic effects of specific DNA alkylators is unknown. We present a 1.7-Å crystal structure of AidB, which bears superficial resemblance to the acyl-CoA dehydrogenase superfamily of flavoproteins. The structure reveals a unique quaternary organization and a distinctive FAD active site that provides a rationale for AidB's limited dehydrogenase activity. A highly electropositive C-terminal domain not present in structural homologs was identified by mutational analysis as the DNA binding site. Structural analysis of the DNA and FAD binding sites provides evidence against AidB-catalyzed DNA repair and supports a model in which AidB acts to prevent alkylation damage by protecting DNA and destroying alkylating agents that have yet to reach their DNA target.

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The lacI Gene as a Target for Mutation in Transgenic Rodents and Escherichia coli

Genetics ◽

10.1093/genetics/148.4.1441 ◽

1998 ◽

Vol 148 (4) ◽

pp. 1441-1451

Author(s):

Johan G de Boer ◽

Barry W Glickman

Keyword(s):

Escherichia Coli ◽

Dna Repair ◽

Dna Binding ◽

Transgenic Animals ◽

Single Copy ◽

Binding Domain ◽

Dna Binding Domain ◽

Large Database ◽

Deletion Mutations ◽

Laci Gene

Abstract The lacI gene has been used extensively for the recovery and analysis of mutations in bacteria with various DNA repair backgrounds and after exposure to a wide variety of mutagens. This has resulted in a large database of information on mutational mechanisms and specificity of many mutagens, as well as the effect of DNA repair background on mutagenicity. Most importantly, knowledge about the mutational sensitivity of the lacI gene is now available, yielding information about mutable nucleotides. This popularity and available knowledge resulted in the use of the lacI gene in transgenic rodents for the study of mutagenesis in mammals, where it resides in ~40 repeated copies. As the number of sequenced mutations recovered from these animals increases, we are able to analyze the sites at which mutations have been recovered in great detail and to compare the recovered sites between bacteria and transgenic animals. The nucleotides that code for the DNA-binding domain are nearly saturated with base substitutions. Even after determining the sequences of ~10,000 mutations recovered from the animals, however, new sites and new changes are still being recovered. In addition, we compare the nature of deletion mutations between bacteria and animals. Based on the nature of deletions in the animals, we conclude that each deletion occurs in a single copy of the gene.

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An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

Molecular and Cellular Biology ◽

10.1128/mcb.14.1.68 ◽

1994 ◽

Vol 14 (1) ◽

pp. 68-76 ◽

Cited By ~ 321

Author(s):

K W Caldecott ◽

C K McKeown ◽

J D Tucker ◽

S Ljungquist ◽

L H Thompson

Keyword(s):

Dna Repair ◽

Affinity Chromatography ◽

Mammalian Cells ◽

Excision Repair ◽

Affinity Purification ◽

Alkylating Agents ◽

Chinese Hamster ◽

Dna Ligase ◽

Base Excision ◽

Dna Ligase Iii

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

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