Identification of pathways controlling DNA damage induced mutation in Saccharomyces cerevisiae

Abstract

Mutation in response to most types of DNA damage is thought to be mediated by the error-prone sub-branch of post-replication repair and the associated translesion synthesis polymerases. To further understand the mutagenic response to DNA damage, we screened a collection of 4848 haploid gene deletion strains of Saccharomyces cerevisiae for decreased damage-induced mutation of the CAN1 gene. Through extensive quantitative validation of the strains identified by the screen, we identified ten genes, which included error-prone post-replication repair genes known to be involved in induced mutation, as well as two additional genes, FYV6 and RNR4. We demonstrate that FYV6 and RNR4 are epistatic with respect to induced mutation, and that they function, at least partially, independently of post-replication repair. This pathway of induced mutation appears to be mediated by an increase in dNTP levels that facilitates lesion bypass by the replicative polymerase Polδ, and it is as important as error-prone post-replication repair in the case of UV-and MMS-induced mutation, but solely responsible for EMS-induced mutation. We show that Rnr4/Polδ-induced mutation is efficiently inhibited by hydroxyurea, a small molecule inhibitor of ribonucleotide reductase, suggesting that if similar pathways exist in human cells, intervention in some forms of mutation may be possible. Keywordsyeast; post-replication repair; induced mutagenesis 1.IntroductionThe stability of the genome is critical for life, but mutation occurs as a result of both exogenous and endogeneous mutagens [1]. Mutations contribute to ageing [2], oncogenesis, tumor progression [3][4][5], and resistance to chemotherapy [6][7][8]. Ironically, because many chemotherapeutic drugs are mutagens, they may themselves play a role in inducing the mutations that give rise to resistance and therapy failure [9][10][11].For many years, it was assumed that mutagens induced DNA damage, and the damaged DNA caused the replicative polymerases to make errors during DNA replication. However, it is now understood that most types of DNA damage inhibit DNA replication, and that the cellular response to this inhibition is critical for the induction of mutation. The cellular response to replication blocks in the yeast Saccharomyces cerevisiae has been intensively studied and is 1Corresponding author Floyd E. Romesberg, Department of Chemistry, The Scripps Research Institute, CB262R, 10550 N. Torrey Pines Rd, La Jolla, CA, 92037, Phone: (858) Fax: (858) 784-7472, E-mail: floyd@scripps.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Pub...

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