2′-Phosphate cyclase activity of RtcA: a potential rationale for the operon organization of RtcA with an RNA repair ligase RtcB in <i>Escherichia coli</i> and other bacterial taxa

Das, Ushati; Shuman, Stewart

doi:10.1261/rna.039917.113

Cited by 40 publications

(65 citation statements)

References 32 publications

(36 reference statements)

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“…D17Rp was converted to D17R>p (Fig. S1A) by reaction with RNA 3′-terminal cyclase as described (22), then recovered by phenol:chloroform extraction and precipitation with ethanol. Quantitative cyclization of the 3′-PO 4 was verified by treating the D17Rp and D17R>p strands with alkaline phosphatase followed by TLC analysis, which showed that all of the 32 P-label in the D17Rp strand was liberated as 32 P i , whereas the label in the D17R>p strand was completely resistant to hydrolysis, as expected for a phosphodiester (22).…”

Section: Methodsmentioning

confidence: 99%

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO ₄ and 5′-OH ends

Das

Chakravarty

Remus

et al. 2013

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

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There are many biological contexts in which DNA damage generates "dirty" breaks with 3′-PO 4 (or cyclic-PO 4 ) and 5′-OH ends that cannot be sealed by DNA ligases. Here we show that the Escherichia coli RNA ligase RtcB can splice these dirty DNA ends via a unique chemical mechanism. RtcB transfers GMP from a covalent RtcB-GMP intermediate to a DNA 3′-PO 4 to form a "capped" 3′ end structure, DNA 3′ pp 5′ G. When a suitable DNA 5′-OH end is available, RtcB catalyzes attack of the 5′-OH on DNA 3′ pp 5′ G to form a 3′-5′ phosphodiester splice junction. Our findings unveil an enzymatic capacity for DNA 3′ capping and the sealing of DNA breaks with 3′-PO 4 and 5′-OH termini, with implications for DNA repair and DNA rearrangements.T he Escherichia coli RtcB is a founding member of a recently discovered family of RNA repair/splicing enzymes that join RNA 2′,3′-cyclic-PO 4 or 3′-PO 4 ends to RNA 5′-OH ends (1-4). RtcB executes a four-step pathway that requires GTP as an energy source and Mn 2+ as a cofactor (5-7). RtcB first reacts with GTP to form a covalent RtcB-(histidinyl 337 -N)-GMP intermediate. It then hydrolyzes the RNA 2′,3′-cyclic-PO 4 end to a 3′-PO 4 and transfers guanylate from His337 to the RNA 3′-PO 4 to form an RNA 3′ pp 5′ G intermediate. Finally, RtcB catalyzes the attack of an RNA 5′-OH on the RNA 3′ pp 5′ G end to form the 3′-5′ phosphodiester splice junction and liberate GMP.The unique chemical mechanism of RtcB overturned a longstanding tenet of nucleic acid enzymology, which held that synthesis of polynucleotide 3′-5′ phosphodiesters proceeds via the attack of a 3′-OH on a high-energy 5′-phosphoanhydride: either a nucleoside 5′-triphosphate in the case of RNA/DNA polymerases or an adenylylated intermediate A 5′ pp 5′ N, in the case of classic RNA/DNA ligases. In light of the wide distribution of RtcB proteins in bacteria, archaea, and metazoa, we raised the prospect of an alternative enzymology based on covalently activated 3′-PO 4 ends (6).In principle, the chemistry of RNA 3′-PO 4 /5′-OH end joining by RtcB might be portable to DNA transactions and pertinent to DNA repair. A variety of hydrolytic nucleases incise the DNA phosphodiester backbone to yield 3′-PO 4 and 5′-OH termini that cannot be joined by DNA ligases. Nonligatable 3′-PO 4 ends are also generated during base excision repair catalyzed by DNA glycosylase/lyase enzymes, during the repair of trapped covalent topoisomerase IB-DNA adducts by tyrosyl-DNA phosphodiesterase 1, and during DNA damage inflicted by ionizing radiation. One way nature solves this "dirty end" problem is by deploying a variety of "end healing" enzymes (8-14). These include 3′-phosphoesterases that convert a 3′-PO 4 to a 3′-OH and 5′-kinases that transform a 5′-OH to a 5′-PO 4 , thereby enabling break sealing by the classic ligase pathway. Given what we now know about RtcB, would it not make sense for nature to also endow a pathway for direct joining of DNA 3′-PO 4 and 5′-OH ends, be it via RtcB or another ligase yet to be discovered?We can extend this thought to DN...

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Section: Methodsmentioning

confidence: 99%

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO ₄ and 5′-OH ends

Das

Chakravarty

Remus

et al. 2013

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

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“…We chose this organism because the putative RNA is not under control of the RtcR transcriptional activator, a feature that prevents its transcription during normal growth in S. Typhimurium and possibly other bacteria Das and Shuman 2013). In M. smegmatis, the predicted YrlA is 5 ′ of the Ro60 ortholog, within the annotated N terminus of the MSMEI_1161 locus (Fig.…”

Section: Bacterial Y Rnas Contain Nucleotide Modifications Characterimentioning

confidence: 99%

Bacterial noncoding Y RNAs are widespread and mimic tRNAs

Chen

Sim

Wurtmann

et al. 2014

RNA

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Many bacteria encode an ortholog of the Ro60 autoantigen, a ring-shaped protein that is bound in animal cells to noncoding RNAs (ncRNAs) called Y RNAs. Studies in Deinococcus radiodurans revealed that Y RNA tethers Ro60 to polynucleotide phosphorylase, specializing this exoribonuclease for structured RNA degradation. Although Ro60 orthologs are present in a wide range of bacteria, Y RNAs have been detected in only two species, making it unclear whether these ncRNAs are common Ro60 partners in bacteria. In this study, we report that likely Y RNAs are encoded near Ro60 in >250 bacterial and phage species. By comparing conserved features, we discovered that at least one Y RNA in each species contains a domain resembling tRNA. We show that these RNAs contain nucleotide modifications characteristic of tRNA and are substrates for several enzymes that recognize tRNAs. Our studies confirm the importance of Y RNAs in bacterial physiology and identify a new class of ncRNAs that mimic tRNA.

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“…A simple way to address this issue is to query whether any or all of the nucleotides of a spliceable polynucleotide substrate for AtRNL (exclusive of the 3 ′ nucleotide) can be replaced by deoxynucleotides. This approach has been fruitful in delineating the minimal RNA content required for other RNA repair enzymes, including T4 RNA ligase 2 (Nandakumar and Shuman 2004), the RNA terminal 2 ′ -O-methyltransferase Hen1 (Jain and Shuman 2011), the RNA 3 ′ -terminal cyclase RtcA (Das and Shuman 2013), and the RNA 3 ′ -PO 4 /5 ′ -OH ligase RtcB (Das et al 2013a). Here, we tested whether AtRNL could splice a HO D17R>p substrate, consisting of 17 deoxynucleotides and a single 32 Plabeled 3 ′ -terminal ribonucleoside-2 ′ ,3 ′ -cyclic-PO 4 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…′ -d(CATATCCGTGTCGCCC T)(rC), to a 5 ′ 32 P-labeled 10-mer DNA strand, 5 ′ -pATTCC GATAG, followed by digestion of the isolated D17RpD10 strand with RNase A to yield HO D17R 3 ′ p, which was then converted to HO D17R>p by RtcA (Das and Shuman 2013).…”

Section: Resultsmentioning

confidence: 99%

Distinctive kinetics and substrate specificities of plant and fungal tRNA ligases

Remus

Shuman

2014

RNA

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Plant and fungal tRNA ligases are trifunctional enzymes that repair RNA breaks with 2 ′ ,3 ′ -cyclic-PO 4 and 5 ′ -OH ends. They are composed of cyclic phosphodiesterase (CPDase) and polynucleotide kinase domains that heal the broken ends to generate the 3 ′ -OH, 2 ′ -PO 4 , and 5 ′ -PO 4 required for sealing by a ligase domain. Here, we use short HO RNA>p substrates to determine, in a one-pot assay format under single-turnover conditions, the order and rates of the CPDase, kinase and ligase steps. The observed reaction sequence for the plant tRNA ligase AtRNL, independent of RNA length, is that the CPDase engages first, converting HO RNA>p to HO RNA 2 ′ p, which is then phosphorylated to pRNA 2 ′ p by the kinase. Whereas the rates of the AtRNL CPDase and kinase reactions are insensitive to RNA length, the rate of the ligase reaction is slowed by a factor of 16 in the transition from 10-mer RNA to 8-mer and further by eightfold in the transition from 8-mer RNA to 6-mer. We report that a single ribonucleoside-2 ′ ,3 ′ -cyclic-PO 4 moiety enables AtRNL to efficiently splice an otherwise all-DNA strand. Our characterization of a fungal tRNA ligase (KlaTrl1) highlights important functional distinctions vis à vis the plant homolog. We find that (1) the KlaTrl1 kinase is 300-fold faster than the AtRNL kinase; and (2) the KlaTrl1 kinase is highly specific for GTP or dGTP as the phosphate donor. Our findings recommend tRNA ligase as a tool to map ribonucleotides embedded in DNA and as a target for antifungal drug discovery.

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2′-Phosphate cyclase activity of RtcA: a potential rationale for the operon organization of RtcA with an RNA repair ligase RtcB in Escherichia coli and other bacterial taxa

Cited by 40 publications

References 32 publications

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO ₄ and 5′-OH ends

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO ₄ and 5′-OH ends

Bacterial noncoding Y RNAs are widespread and mimic tRNAs

Distinctive kinetics and substrate specificities of plant and fungal tRNA ligases

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2′-Phosphate cyclase activity of RtcA: a potential rationale for the operon organization of RtcA with an RNA repair ligase RtcB in Escherichia coli and other bacterial taxa

Cited by 40 publications

References 32 publications

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO 4 and 5′-OH ends

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO 4 and 5′-OH ends

Bacterial noncoding Y RNAs are widespread and mimic tRNAs

Distinctive kinetics and substrate specificities of plant and fungal tRNA ligases

Contact Info

Product

Resources

About

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO ₄ and 5′-OH ends

Rewriting the rules for end joining via enzymatic splicing of DNA 3′-PO ₄ and 5′-OH ends