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Siz2 Prevents Ribosomal DNA Recombination by Modulating Levels of Tof2 in Saccharomyces cerevisiae.

ABSTRACT: Ribosomal DNA (rDNA) recombination in budding yeast is regulated by multiple converging processes, including posttranslational modifications such as SUMOylation. In this study, we report that the absence of a SUMO E3 ligase, Siz2, results in increased unequal rDNA exchange. We show that Siz2 is enriched at the replication fork barrier (RFB) in the rDNA and also controls the homeostasis of Tof2 protein. siz2? resulted in increased accumulation of total Tof2 in the cell and a consequent increase in the enrichment of Tof2 at the rDNA. Overproducing Tof2 ectopically or conditional overexpression of Tof2 also resulted in higher levels of rDNA recombination, suggesting a direct role for Tof2. Additionally, our chromatin immunoprecipitation (ChIP) data indicate that the accumulation of Tof2 in a siz2? mutant resulted in an enhanced association of Fob1, an RFB binding protein at the rDNA at the RFB. This increased Fob1 association at the RFB may have resulted in the elevated rDNA recombination. Our study thus demonstrates that the Tof2 levels modulate recombination at the rDNA.IMPORTANCE The genes that encode rRNA in Saccharomyces cerevisiae are organized as multiple repeats. The repetitive nature and heavy transcription of this region make it prone to DNA breaks. DNA breaks could lead to recombination, which could result in either loss or gain of repeats with detrimental consequences to the cell. Multiple mechanisms operate to maintain the stability of rDNA. Earlier studies reported that the absence of Ulp2, a deSUMOylase, resulted in declining levels of Tof2 and thereby disrupted rDNA silencing. In contrast, our findings suggest that accumulation of Tof2 can also result in increased rDNA recombination, through a mechanism that involves Fob1, an RFB-bound protein. While our study has examined only Tof2, rDNA recombination could be regulated by other proteins through a mechanism similar to this.

SUBMITTER: Abraham NM

PROVIDER: S-EPMC6881720 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Siz2 Prevents Ribosomal DNA Recombination by Modulating Levels of Tof2 in Saccharomyces cerevisiae.

Abraham Neethu Maria NM Ramalingam Kathirvel K Murthy Saketh S Mishra Krishnaveni K

mSphere 20191127 6

Ribosomal DNA (rDNA) recombination in budding yeast is regulated by multiple converging processes, including posttranslational modifications such as SUMOylation. In this study, we report that the absence of a SUMO E3 ligase, Siz2, results in increased unequal rDNA exchange. We show that Siz2 is enriched at the replication fork barrier (RFB) in the rDNA and also controls the homeostasis of Tof2 protein. <i>siz2Δ</i> resulted in increased accumulation of total Tof2 in the cell and a consequent inc ...[more]

PMID: 31776241

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Project description:Nuclease based genome editing systems have emerged as powerful tools to drive genomic alterations and enhance genome evolution via precise engineering in the various human and microbial cells. However, error-prone DNA repair has not been well studied previously to generate diverse genomic alterations and novel phenotypes. Here, we systematically investigated the potential interplay between DNA double strand break (DSB) repair and genome editing tools, and found that modulating the DSB end resection proteins could significantly improve mutational efficiency and diversity without exogenous DNA template in yeast. Deleting SAE2, EXO1, or FUN30, or overexpressing MRE11-H125N (nuclease-dead allele of MRE11), for DSB end resection markedly increased the efficiency of CRISPR/SpCas9 (more than 22-fold) and CRISPR/AsCpf1 (more than 30-fold)-induced mutagenesis. Deleting SAE2 or overexpressing MRE11-H125N substantially diversified CRISPR/SpCas9 or AsCpf1-induced mutation 2-3-fold at URA3 locus, and 3-5-fold at ADE2 locus. Thus, the error-prone DNA repair protein was employed to develop a novel mutagenic genome editing (mGE) strategy, which can increase the mutation numbers and effectively improve the ethanol/glycerol ratio of Saccharomyces cerevisiae through modulating the expression of FPS1 and GPD1. This study highlighted the feasibility of potentially reshaping the capability of genome editing by regulating the different DSB repair proteins and can thus expand the application of genome editing in diversifying gene expression and enhancing genome evolution. IMPORTANCE Most of the published papers about nuclease-assisted genome editing focused on precision engineering in human cells. However, the topic of inducing mutagenesis via error-prone repair has often been ignored in yeast. In this study, we reported that perturbing DNA repair, especially modifications of the various DSB end resection-related proteins, could greatly improve the mutational efficiency and diversity, and thus functionally reshape the capability of the different genome editing tools without requiring an exogenous DNA template in yeast. Specifically, mutagenic genome editing (mGE) was developed based on CRISPR/AsCpf1 and MRE11-H125N overexpression, and used to generate promoters of different strengths more efficiently. Thus, this work provides a novel method to diversify gene expression and enhance genome evolution.

Dataset Information

Siz2 Prevents Ribosomal DNA Recombination by Modulating Levels of Tof2 in Saccharomyces cerevisiae.

Publications

Siz2 Prevents Ribosomal DNA Recombination by Modulating Levels of Tof2 in Saccharomyces cerevisiae.

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