RESUMO
RNA-DNA hybrids form throughout the chromosome during normal growth and under stress conditions. When left unresolved, RNA-DNA hybrids can slow replication fork progression, cause DNA breaks, and increase mutagenesis. To remove hybrids, all organisms use ribonuclease H (RNase H) to specifically degrade the RNA portion. Here we show that, in addition to chromosomally encoded RNase HII and RNase HIII, Bacillus subtilis NCIB 3610 encodes a previously uncharacterized RNase HI protein, RnhP, on the endogenous plasmid pBS32. Like other RNase HI enzymes, RnhP incises Okazaki fragments, ribopatches, and a complementary RNA-DNA hybrid. We show that while chromosomally encoded RNase HIII is required for pBS32 hyper-replication, RnhP compensates for the loss of RNase HIII activity on the chromosome. Consequently, loss of RnhP and RNase HIII impairs bacterial growth. We show that the decreased growth rate can be explained by laggard replication fork progression near the terminus region of the right replichore, resulting in SOS induction and inhibition of cell division. We conclude that all three functional RNase H enzymes are present in B. subtilis NCIB 3610 and that the plasmid-encoded RNase HI contributes to chromosome stability, while the chromosomally encoded RNase HIII is important for chromosome stability and plasmid hyper-replication.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Ribonuclease H/metabolismo , Sequência de Aminoácidos/genética , DNA/genética , Replicação do DNA/genética , Instabilidade Genômica/genética , Plasmídeos/genética , RNA/metabolismo , Ribonuclease H/genética , Especificidade por Substrato/genéticaRESUMO
Oxazolidinones are promising candidates for the treatment of Mycobacterium tuberculosis infections. We isolated linezolid-resistant strains from H37Rv (Euro-American) and HN878 (East-Asian) strains; resistance frequencies were similar in the two strains. Mutations were identified in ribosomal protein L3 (RplC) and the 23S rRNA (rrl). All mutant strains were cross resistant to sutezolid; a subset was cross resistant to chloramphenicol. Mutations in rrl led to growth impairment and decreased fitness that may limit spread in clinical settings.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Linezolida/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/genética , Inibidores da Síntese de Proteínas/farmacologia , RNA Ribossômico 23S/genética , Proteínas Ribossômicas/genética , Antituberculosos/farmacologia , Sequência de Bases , Sítios de Ligação , Cloranfenicol/farmacologia , DNA Bacteriano/genética , Humanos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/isolamento & purificação , Oxazolidinonas/farmacologia , Proteína Ribossômica L3 , Análise de Sequência de DNA , Tuberculose Pulmonar/tratamento farmacológico , Tuberculose Pulmonar/microbiologiaRESUMO
Degrons are minimal protein features that are sufficient to target proteins for degradation. In most cases, degrons allow recognition by components of the cytosolic ubiquitin proteasome system. Currently, all of the identified degrons only function within the cytosol. Using Saccharomyces cerevisiae, we identified the first short linear sequences that function as degrons from the endoplasmic reticulum (ER) lumen. We show that when these degrons are transferred to proteins, they facilitate proteasomal degradation through the ERAD system. These degrons enable degradation of both luminal and integral membrane ER proteins, expanding the types of proteins that can be targeted for degradation in budding yeast and mammalian tissue culture. This discovery provides a framework to target proteins for degradation from the previously unreachable ER lumen and builds toward therapeutic approaches that exploit the highly-conserved ERAD system.