Recombination is responsible for the increased recovery of drug-resistant mutants with hypermutated genomes in resting Yeast Diploids Expressing APOBEC deaminases

Artem G. Lada, Elena I. Stepchenkova, Anna S. Zhuk, Sergei F. Kliver, Igor B. Rogozin, Dmitrii E. Polev, Alok Dhar, Youri I Pavlov

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

DNA editing deaminases (APOBECs) are implicated in generation of mutations in somatic cells during tumorigenesis. APOBEC-dependent mutagenesis is thought to occur during transient exposure of unprotected single-stranded DNA. Mutations frequently occur in clusters (kataegis). We investigated mechanisms of mutant generation in growing and resting diploid yeast expressing APOBEC from sea lamprey, PmCDA1, whose kataegistic effect was previously shown to be associated with transcription. We have found that the frequency of canavanine-resistant mutants kept raising after growth cessation, while the profile of transcription remained unchanged. Surprisingly, the overall number of mutations in the genomes did not elevate in resting cells. Thus, mutations were accumulated during vigorous growth stage with both intense replication and transcription. We found that the elevated recovery of can1 mutant clones in non-growing cells is the result of loss of heterozygosity (LOH) leading to clusters of homozygous mutations in the chromosomal regions distal to the reporter gene. We confirmed that recombination frequency in resting cells was elevated by orders of magnitude, suggesting that cells were transiently committed to meiotic levels of recombination, a process referred to in yeast genetics as return-to-growth. In its extreme, on day 6 of starvation, a few mutant clones were haploid, likely resulting from completed meiosis. Distribution of mutations along chromosomes indicated that PmCDA1 was active during ongoing recombination events and sometimes produced characteristic kataegis near initial breakpoints. AID and APOBEC1 behaved similar to PmCDA1. We conclude that replication, transcription, and mitotic recombination contribute to the recovered APOBEC-induced mutations in resting diploids. The mechanism is relevant to the initial stages of oncogenic transformation in terminally differentiated cells, when recombination may lead to the LOH exposing recessive mutations induced by APOBECs in cell's history and to acquisition of new mutations near original break.

Original languageEnglish (US)
Article number202
JournalFrontiers in Genetics
Volume8
Issue numberDEC
DOIs
StatePublished - Dec 12 2017

Fingerprint

Diploidy
Genetic Recombination
Yeasts
Genome
Mutation
Pharmaceutical Preparations
Loss of Heterozygosity
Growth
Clone Cells
Petromyzon
Canavanine
APOBEC Deaminases
Single-Stranded DNA
Haploidy
Meiosis
Starvation
Reporter Genes
Mutagenesis
Carcinogenesis
Chromosomes

Keywords

  • APOBECs
  • Kataegis
  • Mutants in resting diploid yeast
  • Next generation sequencing
  • Recombination
  • Tumorigenesis

ASJC Scopus subject areas

  • Molecular Medicine
  • Genetics
  • Genetics(clinical)

Cite this

Recombination is responsible for the increased recovery of drug-resistant mutants with hypermutated genomes in resting Yeast Diploids Expressing APOBEC deaminases. / Lada, Artem G.; Stepchenkova, Elena I.; Zhuk, Anna S.; Kliver, Sergei F.; Rogozin, Igor B.; Polev, Dmitrii E.; Dhar, Alok; Pavlov, Youri I.

In: Frontiers in Genetics, Vol. 8, No. DEC, 202, 12.12.2017.

Research output: Contribution to journalArticle

Lada, Artem G. ; Stepchenkova, Elena I. ; Zhuk, Anna S. ; Kliver, Sergei F. ; Rogozin, Igor B. ; Polev, Dmitrii E. ; Dhar, Alok ; Pavlov, Youri I. / Recombination is responsible for the increased recovery of drug-resistant mutants with hypermutated genomes in resting Yeast Diploids Expressing APOBEC deaminases. In: Frontiers in Genetics. 2017 ; Vol. 8, No. DEC.
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AU - Lada, Artem G.

AU - Stepchenkova, Elena I.

AU - Zhuk, Anna S.

AU - Kliver, Sergei F.

AU - Rogozin, Igor B.

AU - Polev, Dmitrii E.

AU - Dhar, Alok

AU - Pavlov, Youri I

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AB - DNA editing deaminases (APOBECs) are implicated in generation of mutations in somatic cells during tumorigenesis. APOBEC-dependent mutagenesis is thought to occur during transient exposure of unprotected single-stranded DNA. Mutations frequently occur in clusters (kataegis). We investigated mechanisms of mutant generation in growing and resting diploid yeast expressing APOBEC from sea lamprey, PmCDA1, whose kataegistic effect was previously shown to be associated with transcription. We have found that the frequency of canavanine-resistant mutants kept raising after growth cessation, while the profile of transcription remained unchanged. Surprisingly, the overall number of mutations in the genomes did not elevate in resting cells. Thus, mutations were accumulated during vigorous growth stage with both intense replication and transcription. We found that the elevated recovery of can1 mutant clones in non-growing cells is the result of loss of heterozygosity (LOH) leading to clusters of homozygous mutations in the chromosomal regions distal to the reporter gene. We confirmed that recombination frequency in resting cells was elevated by orders of magnitude, suggesting that cells were transiently committed to meiotic levels of recombination, a process referred to in yeast genetics as return-to-growth. In its extreme, on day 6 of starvation, a few mutant clones were haploid, likely resulting from completed meiosis. Distribution of mutations along chromosomes indicated that PmCDA1 was active during ongoing recombination events and sometimes produced characteristic kataegis near initial breakpoints. AID and APOBEC1 behaved similar to PmCDA1. We conclude that replication, transcription, and mitotic recombination contribute to the recovered APOBEC-induced mutations in resting diploids. The mechanism is relevant to the initial stages of oncogenic transformation in terminally differentiated cells, when recombination may lead to the LOH exposing recessive mutations induced by APOBECs in cell's history and to acquisition of new mutations near original break.

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