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Use of yeast transformation by oligonucleotides to study DNA lesion bypass in vivo

Chie Otsuka1, Keita Kobayashi, Naho Kawaguchi

  • 1Gene Research Center, Okayama University, Tsushima, Okayama, Japan.

Mutation Research
|May 9, 2002
PubMed

Insights

This study investigated DNA lesion mutagenicity in yeast. The nucleoside analog dP showed miscoding potential, while UV photoproducts required the REV1 gene for bypass, revealing distinct DNA repair pathways.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • DNA lesions are alterations to DNA structure that can lead to mutations.
  • Understanding mutagenic specificities of DNA lesions is crucial for comprehending genome stability and disease mechanisms.
  • Yeast models provide a powerful system for studying DNA repair and mutagenesis in vivo.

Purpose of the Study:

  • To investigate the in vivo mutagenic specificities of two distinct DNA lesions: a nucleoside analog (dP) and a UV-induced photoproduct.
  • To elucidate the role of the REV1 gene in the bypass of DNA lesions during replication.
  • To characterize the DNA polymerase templating properties and mutation outcomes of these lesions in a yeast CYC1 oligonucleotide transformation assay.

Main Methods:

  • Oligonucleotide-directed yeast transformation assay using cyc1 mutants.
  • Introduction of a nucleoside analog (dP) and a TT pyrimidine(6-4)pyrimidone photoproduct into DNA oligonucleotides.
  • Analysis of transforming activity and sequencing of resulting transformants to determine mutation frequencies and specificities.

Main Results:

  • The dP lesion exhibited similar transforming activity to unmodified oligonucleotides, suggesting it acts as a good template.
  • dP directed the in vivo incorporation of guanine and adenine at a ratio of at least 1:20.
  • The TT photoproduct significantly reduced transforming activity (3-5%) and its bypass was absolutely dependent on the REV1 gene.
  • Sequence analysis revealed the photoproduct was read as TT and TC in a 3:7 ratio, indicating high mutagenicity.

Conclusions:

  • The nucleoside analog dP demonstrates miscoding potential in vivo, primarily incorporating adenine opposite guanine.
  • The TT pyrimidine(6-4)pyrimidone photoproduct is a strong replication-blocking lesion whose bypass is critically dependent on the REV1 gene.
  • These findings highlight distinct mechanisms for processing different types of DNA lesions in yeast, involving specific genes like REV1 for error-prone bypass.

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