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This study examines the contributions of ribonucleases (RNase)and human genes to hybrid removal, mutagenesis, and DNA replication genome-wide, revealing distinct patterns of mutagenesis and hybrid accumulation in cells lacking these ribonucleases. The accumulation of hybrids is found to be influenced by gene expression levels, and disruptions in DNA replication in certain cells lead to transversions and structural variations.

The budding yeast Saccharomyces cerevisiae undergoes a stress-responsive transition to a pseudohyphal growth form in which cells elongate and remain connected in multicellular filaments. Pseudohyphal growth is regulated through conserved signaling networks that control cell growth and the response to glucose or nitrogen limitation in metazoans. These networks are incompletely understood, and our studies identify the TORC1- and PKA-regulated kinase Ksp1p as a key stress-responsive signaling effector in the yeast pseudohyphal growth response. The kinase-defective ksp1-K47D allele results in decreased pseudohyphal morphology at the cellular and colony level, indicating that Ksp1p kinase signaling is required for pseudohyphal filamentation. To determine the functional consequences of Ksp1p signaling, we implemented transcriptional profiling and quantitative phosphoproteomic analysis of ksp1-K47D on a global scale. Ksp1p kinase signaling maintains wild-type transcript levels of many pathways for amino acid synthesis and metabolism, relevant for the regulation of translation under conditions of nutrient stress. Proteins in stress-responsive ribonucleoprotein granules are regulated post-translationally by Ksp1p, and the Ksp1p-dependent phosphorylation sites S176 in eIF4G/Tif4631p and S436 in Pbp1p are required for wild-type levels of pseudohyphal growth and Protein Kinase A pathway activity. Pbp1p and Tif4631p localize in stress granules, and the ksp1 null mutant shows elevated abundance of Pbp1p puncta relative to wild-type. Collectively, the Ksp1p kinase signaling network integrates polarized pseudohyphal morphogenesis and translational regulation through the stressresponsive transcriptional control of pathways for amino acid metabolism and post-translational modification of translation factors affecting stress granule abundance. [ABSTRACT FROM AUTHOR]

DNA replication is regulated by factors that promote or inhibit initiation. In Bacillus subtilis, YabA is a negative regulator of DNA replication initiation while the newly identified kinase CcrZ is a positive regulator. The consequences of under-initiation or over-initiation of DNA replication to genome stability remain unclear. In this work, we measure origin to terminus ratios as a proxy for replication initiation activity. We show that Δ ccrZ and several ccrZ alleles under-initiate DNA replication while ablation of yabA or overproduction of CcrZ leads to over-initiation. We find that cells under-initiating DNA replication have few incidents of replication fork stress as determined by low formation of RecA-GFP foci compared with wild type. In contrast, cells over-initiating DNA replication show levels of RecA-GFP foci formation analogous to cells directly challenged with DNA damaging agents. We show that cells under-initiating and over-initiating DNA replication were both sensitive to mitomycin C and that changes in replication initiation frequency cause increased sensitivity to genotoxic stress. With these results, we propose that cells under-initiating DNA replication are sensitive to DNA damage due to a shortage of DNA for repair through homologous recombination. For cells over-initiating DNA replication, we propose that an increase in the number of replication forks leads to replication fork stress which is further exacerbated by chromosomal DNA damage. Together, our study shows that DNA replication initiation frequency must be tightly controlled as changes in initiation influence replication fork fate and the capacity of cells to efficiently repair damage to their genetic material.

Bacillus subtilis is a widely studied Gram-positive bacterium that serves as an important model for understanding processes critical for several areas of biology including biotechnology and human health. B. subtilis has several advantages as a model organism: it is easily grown under laboratory conditions, it has a rapid doubling time, it is relatively inexpensive to maintain, and it is nonpathogenic. Over the last 50 years, advancements in genetic engineering have continued to make B. subtilis a genetic workhorse in scientific discovery. In this chapter, we describe methods for traditional gene disruptions, use of gene deletion libraries from the Bacillus Genetic Stock Center, allelic exchange, CRISPRi, and CRISPR/Cas9. Additionally, we provide general materials and equipment needed, strengths and limitations, time considerations, and troubleshooting notes to perform each method. Use of the methods outlined in this chapter will allow researchers to create gene insertions, deletions, substitutions, and RNA interference strains through a variety of methods custom to each application., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)