The nuclease activities of both the <scp>S</scp>mr domain and an additional <scp>LDLK</scp> motif are required for an efficient anti‐recombination function of <scp><i>H</i></scp><i>elicobacter pylori</i> <scp>MutS2</scp>

Damke, Prashant P.; Dhanaraju, Rajkumar; Marsin, Stéphanie; Radicella, J. Pablo; Rao, Desirazu N.

doi:10.1111/mmi.13003

Cited by 19 publications

(33 citation statements)

References 64 publications

(121 reference statements)

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“…2 B and C), which is consistent with the DNA endonuclease activity observed in other SMR-containing proteins (20)(21)(22)(23)(24)(25)(26). In addition, our results showed that the SMR domain of SOT1 has RNA endonuclease activity (Fig.…”

Section: Discussionsupporting

confidence: 90%

“…Because SMR domains typically have DNA nicking activity (20)(21)(22)(23)(24)(25)(26), we investigated whether the SMR domain of SOT1 retained the ability to nick supercoiled DNA. We found that recombinant SMR protein SOT1 SMR (amino acids 603-710 of SOT1) cleaved supercoiled pUC19 to open circular and linear conformations and that the metal ions Mn 2+ and Mg 2+ increased the DNA endonuclease activity of SOT1 SMR ( Fig.…”

Section: Significancementioning

confidence: 99%

“…SMR proteins are widely distributed in almost all organisms (19). Recent studies demonstrated the SMR domain exhibits DNA nicking nuclease activity in vitro (20)(21)(22)(23)(24)(25). Furthermore, a C-terminal SMR domain in Leishmania donovani S-phase mRNA cycling sequence binding protein (CSBP) has RNA cleavage activity in vitro (26).…”

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confidence: 99%

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PPR-SMR protein SOT1 has RNA endonuclease activity

Zhou

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Numerous attempts have been made to identify and engineer sequence-specific RNA endonucleases, as these would allow for efficient RNA manipulation. However, no natural RNA endonuclease that recognizes RNA in a sequence-specific manner has been described to date. Here, we report that SUPPRESSOR OF THYLAKOID FORMATION 1 (SOT1), an Arabidopsis pentatricopeptide repeat (PPR) protein with a small MutS-related (SMR) domain, has RNA endonuclease activity. We show that the SMR moiety of SOT1 performs the endonucleolytic maturation of 23S and 4.5S rRNA through the PPR domain, specifically recognizing a 13-nucleotide RNA sequence in the 5′ end of the chloroplast 23S-4.5S rRNA precursor. In addition, we successfully engineered the SOT1 protein with altered PPR motifs to recognize and cleave a predicted RNA substrate. Our findings point to SOT1 as an exciting tool for RNA manipulation.photosynthesis | PPR-SMR protein | RNA endonuclease | rRNA biogenesis S equence-specific RNA endonucleases are crucial to establishing RNA manipulation technology (1). Compared with DNA editing, RNA manipulation could be more useful and reversible because it does not result in permanent changes to the genome. In addition, sequence-specific RNA endonucleases could potentially be used as an RNA silencing tool to complement RNAi, because RNAi is sometimes ineffective in certain organisms and RNAi machinery is not present in cellular compartments such as chloroplasts and mitochondria. Despite extensive investigations, a natural RNA endonuclease that recognizes RNA in an intrinsic sequence-specific manner has not yet been identified.Pentatricopeptide repeat (PPR) proteins exist in eukaryotes, have greatly expanded in terrestrial plants, and take part in most RNA metabolic processes in organelles (2-5). The PPR domain can specifically recognize RNAs in an intrinsic sequence-specific manner (5-8). The 2nd, 5th, and 35th (or 1st, 4th, and 34th or 3rd, 6th, and 1st in other numbering systems) residues at each repeat are considered to be RNA selection "codes" (9-11). Based on these codes, several PPR proteins have been successfully modified to recognize predictable RNA targets (9,(12)(13)(14)(15)(16)(17).The small MutS-related (SMR) domain was originally identified at the C terminus of MutS2 in the cyanobacterium Synechocystis (18). SMR proteins are widely distributed in almost all organisms (19). Recent studies demonstrated the SMR domain exhibits DNA nicking nuclease activity in vitro (20-25). Furthermore, a C-terminal SMR domain in Leishmania donovani S-phase mRNA cycling sequence binding protein (CSBP) has RNA cleavage activity in vitro (26). These findings suggest that the SMR domain has nuclease activity.Interestingly, a small protein family containing both PPR and SMR domains was recently described (27). PPR-SMR proteins are found mainly in land plants. Arabidopsis thaliana contains eight PPR-SMR proteins localized to the organelles, including mitochondria and chloroplasts (27). Currently, four PPR-SMR proteins have been characterized. They play ...

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Section: Discussionsupporting

confidence: 90%

Section: Significancementioning

confidence: 99%

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PPR-SMR protein SOT1 has RNA endonuclease activity

Zhou

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…HpMutS2 has been shown to have ATPase activity that is stimulated by a four-way junction (Holliday junction) and by a fork or "Y" structure (19). A more recent study found that HpMutS2 has nuclease activity toward both single-stranded DNA and Holliday junctions that is dependent on two nuclease sites in the protein: the Smr domain and an N-terminal LDLK motif (45). Interestingly, the LDLK motif is not conserved in B. subtilis or T. thermophilus.…”

Section: Discussionmentioning

confidence: 99%

“…Taken together, these results suggest that during repair of MMC adducts, MutS2 contributes to a step that is recA dependent and that MutS2 does not participate in a step of MMC repair involving NER. Given that MutS2 proteins in other organisms have been shown to bind Holliday junctions (19,22,45), we chose to test whether there was a genetic interaction with the primary Holliday junction endonuclease RecU (46). To this end, we tested a strain either lacking recU (ΔrecU::erm) or lacking both mutS2 and recU in the plating efficiency assay.…”

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confidence: 99%

MutS2 Promotes Homologous Recombination in Bacillus subtilis

Burby

Simmons

2017

J Bacteriol

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Bacterial MutS proteins are subdivided into two families, MutS1 and MutS2. MutS1 family members recognize DNA replication errors during their participation in the well-characterized mismatch repair (MMR) pathway. In contrast to the well-described function of MutS1, the function of MutS2 in bacteria has remained less clear. In Helicobacter pylori and Thermus thermophilus, MutS2 has been shown to suppress homologous recombination. The role of MutS2 is unknown in the Grampositive bacterium Bacillus subtilis. In this work, we investigated the contribution of MutS2 to maintaining genome integrity in B. subtilis. We found that deletion of mutS2 renders B. subtilis sensitive to the natural antibiotic mitomycin C (MMC), which requires homologous recombination for repair. We demonstrate that the C-terminal small MutS-related (Smr) domain is necessary but not sufficient for tolerance to MMC. Further, we developed a CRISPR/Cas9 genome editing system to test if the inducible prophage PBSX was the underlying cause of the observed MMC sensitivity. Genetic analysis revealed that MMC sensitivity was dependent on recombination and not on nucleotide excision repair or a symptom of prophage PBSX replication and cell lysis. We found that deletion of mutS2 resulted in decreased transformation efficiency using both plasmid and chromosomal DNA. Further, deletion of mutS2 in a strain lacking the Holliday junction endonuclease gene recU resulted in increased MMC sensitivity and decreased transformation efficiency, suggesting that MutS2 could function redundantly with RecU. Together, our results support a model where B. subtilis MutS2 helps to promote homologous recombination, demonstrating a new function for bacterial MutS2.IMPORTANCE Cells contain pathways that promote or inhibit recombination. MutS2 homologs are Smr-endonuclease domain-containing proteins that have been shown to function in antirecombination in some bacteria. We present evidence that B. subtilis MutS2 promotes recombination, providing a new function for MutS2. We found that cells lacking mutS2 are sensitive to DNA damage that requires homologous recombination for repair and have reduced transformation efficiency. Further analysis indicates that the C-terminal Smr domain requires the N-terminal portion of MutS2 for function in vivo. Moreover, we show that a mutS2 deletion is additive with a recU deletion, suggesting that these proteins have a redundant function in homologous recombination. Together, our study shows that MutS2 proteins have adapted different functions that impact recombination.

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The GIY‐YIG endonuclease domain of Arabidopsis MutS homolog 1 specifically binds to branched DNA structures

et al. 2018

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In plant organelle genomes, homeologous recombination between heteroallelic positions of repetitive sequences is increased by dysfunction of the gene encoding MutS homolog 1 (MSH1), a plant organelle‐specific homolog of bacterial mismatch‐binding protein MutS1. The C‐terminal region of plant MSH1 contains the GIY‐YIG endonuclease motif. The biochemical characteristics of plant MSH1 have not been investigated; accordingly, the molecular mechanism by which plant MSH1 suppresses homeologous recombination is unknown. Here, we characterized the recombinant GIY‐YIG domain of Arabidopsis thaliana MSH1, showing that the domain possesses branched DNA‐specific DNA‐binding activity. Interestingly, the domain exhibited no endonuclease activity, suggesting that the mismatch‐binding domain is required for DNA incision. Based on these results, we propose a possible mechanism for MSH1‐dependent suppression of homeologous recombination.

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The nuclease activities of both the Smr domain and an additional LDLK motif are required for an efficient anti‐recombination function of Helicobacter pylori MutS2

Cited by 19 publications

References 64 publications

PPR-SMR protein SOT1 has RNA endonuclease activity

PPR-SMR protein SOT1 has RNA endonuclease activity

MutS2 Promotes Homologous Recombination in Bacillus subtilis

The GIY‐YIG endonuclease domain of Arabidopsis MutS homolog 1 specifically binds to branched DNA structures

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