Biochemical analysis of UV mutagenesis in Escherichia coli by using a cell-free reaction coupled to a bioassay: identification of a DNA repair-dependent, replication-independent pathway.

Biochemical analysis of UV mutagenesis in Escherichia coli by using a cell-free reaction coupled to a bioassay: identification of a DNA repair-dependent, replication-independent pathway. Research Abstract Details 

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Biochemical analysis of UV mutagenesis in Escherichia coli by using a cell-free reaction coupled to a bioassay: identification of a DNA repair-dependent, replication-independent pathway. Abstract Text:

Incubation of UV-irradiated plasmid DNA with a protein extract prepared from Escherichia coli cells led to the production of mutations in the cro gene residing on the plasmid. The mutations were detected in a subsequent bioassay step, which involved transformation of an indicator strain with the plasmid DNA that was retrieved from the reaction mixture, followed by plating on lactose/MacConkey plates. UV mutations produced in this cell-free reaction required the recA and umuC gene products and were prevented by rifampicin, an inhibitor of RNA polymerase, which inhibited plasmid replication. Removal of pyrimidine photodimers from the plasmid by enzymatic photoreactivation after the in vitro stage, but prior to transformation, increased plasmid survival as expected. Surprisingly, it also caused a large increase in the frequency of UV mutations detected in the bioassay. This photoreactivation-stimulated in vitro UV mutagenesis was dependent on the excision repair genes uvrA, uvrB, and uvrC and occurred in the absence of DNA replication. This suggests that two distinct UV mutagenesis pathways occurred in vitro: a replication-dependent pathway (type I) and a repair-dependent pathway (type II). DNA sequence analysis of type II UV mutations revealed a spectrum similar to that of in vivo UV mutagenesis. When the photoreactivation step was included in the protocol, type II UV mutagenesis did not require the RecA and UmuC proteins. These results are in agreement with the in vivo delayed photoreactivation phenomenon, where the removal of photodimers after an incubation period eliminated the requirement for RecA and UmuC in UV mutagenesis. The above system will enable the biochemical analysis of UV mutagenesis and the isolation of proteins involved in the process.

Biochemical analysis of UV mutagenesis in Escherichia coli by using a cell-free reaction coupled to a bioassay: identification of a DNA repair-dependent, replication-independent pathway. Publishing Authors By Initials

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Biochemical analysis of UV mutagenesis in Escherichia coli by using a cell-free reaction coupled to a bioassay: identification of a DNA repair-dependent, replication-independent pathway. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Proceedings of the National Academy of Sciences of

VOLUME: 89

Page Numbers: 3300-4

Journal Abbreviation: Proc. Natl. Acad. Sci. U.S.A.

ISSN: 0027-8424

DAY: 15

MONTH: Apr

YEAR: 1992

Biochemical analysis of UV mutagenesis in Escherichia coli by using a cell-free reaction coupled to a bioassay: identification of a DNA repair-dependent, replication-independent pathway. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 7505876

Biochemical analysis of UV mutagenesis in Escherichia coli by using a cell-free reaction coupled to a bioassay: identification of a DNA repair-dependent, replication-independent pathway. Keywords Mesh Terms:

KEYWORDS: Viral Regulatory and Accessory Proteins

MESH TERMS: genetics

Chemical & Substance for Abstract: Biochemical analysis of UV mutagenesis in Escherichia coli by using a cell-free reaction coupled to a bioassay: identification of a DNA repair-dependent, replication-independent pathway. Information

Substance Name: Rec A Recombinases

Registry Number: EC 2.7.7.-

Grant and Affiliation Information for Biochemical analysis of UV mutagenesis in Escherichia coli by using a cell-free reaction coupled to a bioassay: identification of a DNA repair-dependent, replication-independent pathway.

AFFILIATION: Department of Biochemistry, Weizmann Institute of Science, Rehovot, Israel.

Country: UNITED STATES

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MEDLINETA: Proc Natl Acad Sci U S A

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