Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases

Zhelkovsky, Alexander; McReynolds, Larry A.

doi:10.1074/jbc.m114.612929

Cited by 13 publications

(18 citation statements)

References 34 publications

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“…Although the physiological substrate and biological function of Rnl3 are not known, it can act to circularize single-stranded RNA and DNA ( 7 , 18 ). Methanobacterium thermoautotrophicum Rnl3 (MthRnl) can also convert 3′-phosphorylated RNA termini into 2′, 3′-cyclic phosphate ( 19 ). Under a non-optimal condition, MthRnl can remove the 5′-adenylate on AppRNA to generate 5′-PO 4 RNA ( 18 ).…”

Section: Introductionmentioning

confidence: 99%

Structural and mutational analysis of archaeal ATP-dependent RNA ligase identifies amino acids required for RNA binding and catalysis

Yoshinari

Ghosh

et al. 2016

Nucleic Acids Res

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An ATP-dependent RNA ligase from Methanobacterium thermoautotrophicum (MthRnl) catalyzes intramolecular ligation of single-stranded RNA to form a closed circular RNA via covalent ligase-AMP and RNA-adenylylate intermediate. Here, we report the X-ray crystal structures of an MthRnl•ATP complex as well as the covalent MthRnl–AMP intermediate. We also performed structure-guided mutational analysis to survey the functions of 36 residues in three component steps of the ligation pathway including ligase-adenylylation (step 1), RNA adenylylation (step 2) and phosphodiester bond synthesis (step 3). Kinetic analysis underscored the importance of motif 1a loop structure in promoting phosphodiester bond synthesis. Alanine substitutions of Thr117 or Arg118 favor the reverse step 2 reaction to deadenylate the 5′-AMP from the RNA-adenylate, thereby inhibiting step 3 reaction. Tyr159, Phe281 and Glu285, which are conserved among archaeal ATP-dependent RNA ligases and are situated on the surface of the enzyme, are required for RNA binding. We propose an RNA binding interface of the MthRnl based on the mutational studies and two sulfate ions that co-crystallized at the active site cleft in the MthRnl–AMP complex.

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Section: Introductionmentioning

confidence: 99%

Structural and mutational analysis of archaeal ATP-dependent RNA ligase identifies amino acids required for RNA binding and catalysis

Yoshinari

Ghosh

et al. 2016

Nucleic Acids Res

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“…Thus, there is considerable plasticity in the direction of the RtcA pathway (2′,3′ cyclization versus 5′ A capping), with attendant uncertainty as to what the ‘real’ substrates of RtcA are. This theme is amplified by the recent report of Zhelkovsky and McReynolds ( 27 ) that certain classic ATP-dependent thermophilic RNA ligases, which join 3′-OH/5′-PO 4 ends via an AppRNA intermediate, are also capable of adenylylating a DNA 3′-PO 4 end to form a stable DNAppA product. In effect, the thermophilic RNA ligases double as DNA 3′ capping enzymes, albeit with an A cap rather than a G cap.…”

Section: Resultsmentioning

confidence: 99%

“…In effect, the thermophilic RNA ligases double as DNA 3′ capping enzymes, albeit with an A cap rather than a G cap. Following on our discovery of aprataxin as a DNAppG 3′ de-capping enzyme, they found that Saccharomyces cerevisiae aprataxin removes the A cap from DNAppA ( 27 ).…”

Section: Resultsmentioning

confidence: 99%

DNA_3′pp_5′G de-capping activity of aprataxin: effect of cap nucleoside analogs and structural basis for guanosine recognition

2015

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DNA3′pp5′G caps synthesized by the 3′-PO4/5′-OH ligase RtcB have a strong impact on enzymatic reactions at DNA 3′-OH ends. Aprataxin, an enzyme that repairs A5′pp5′DNA ends formed during abortive ligation by classic 3′-OH/5′-PO4 ligases, is also a DNA 3′ de-capping enzyme, converting DNAppG to DNA3′p and GMP. By taking advantage of RtcB's ability to utilize certain GTP analogs to synthesize DNAppN caps, we show that aprataxin hydrolyzes inosine and 6-O-methylguanosine caps, but is not adept at removing a deoxyguanosine cap. We report a 1.5 Å crystal structure of aprataxin in a complex with GMP, which reveals that: (i) GMP binds at the same position and in the same anti nucleoside conformation as AMP; and (ii) aprataxin makes more extensive nucleobase contacts with guanine than with adenine, via a hydrogen bonding network to the guanine O6, N1, N2 base edge. Alanine mutations of catalytic residues His147 and His149 abolish DNAppG de-capping activity, suggesting that the 3′ de-guanylylation and 5′ de-adenylylation reactions follow the same pathway of nucleotidyl transfer through a covalent aprataxin-(His147)–NMP intermediate. Alanine mutation of Asp63, which coordinates the guanosine ribose hydroxyls, impairs DNAppG de-capping.

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“…Initially described as an adenylate decapping enzyme acting on abortive AppDNA intermediates formed by ATP-dependent DNA ligases, aprataxin was subsequently shown to have 3= decapping activity on a DNAppG end formed by E. coli RtcB (12,24) and on DNAppA ends generated by an ATP-dependent thermophilic RNA ligase (38). Here, we extend the aprataxin repertoire by showing that it can remove a 5= guanylate cap from GppDNA formed by M. xanthus RtcB3.…”

Section: Discussionmentioning

confidence: 96%

“…Thus, there is considerable plasticity in the direction of the RtcA pathway (2=,3= cyclization versus 5= A capping), with resulting uncertainty as to what the real substrates of RtcA are. This theme was amplified by the report that certain classic ATP-dependent thermophilic RNA ligases, which join 3=-OH/5=-PO 4 ends via an AppRNA intermediate, are also capable of adenylylating a DNA 3=-PO 4 end to form a stable DNAppA product (38). In effect, the 32 P-labeled 24-mer, 12-mer, or 6-mer pDNA OH substrates or 0.1 M 5= 32 P-labeled 12-mer pRNA OH substrate (with nucleotide sequences as shown), and 1 M RtcB3 were incubated at 30°C.…”

Section: Discussionmentioning

confidence: 99%

Characterization of 3′-Phosphate RNA Ligase Paralogs RtcB1, RtcB2, and RtcB3 from Myxococcus xanthus Highlights DNA and RNA 5′-Phosphate Capping Activity of RtcB3

Maughan

Shuman

2015

J Bacteriol

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Escherichia coli RtcB exemplifies a family of GTP-dependent RNA repair/splicing enzymes that join 3=-PO 4 ends to 5=-OH ends via stable RtcB-(histidinyl-N)-GMP and transient RNA 3= pp 5= G intermediates. E. coli RtcB also transfers GMP to a DNA 3=-PO 4 end to form a stable "capped" product, DNA 3= pp 5= G. RtcB homologs are found in a multitude of bacterial proteomes, and many bacteria have genes encoding two or more RtcB paralogs; an extreme example is Myxococcus xanthus, which has six RtcBs. In this study, we purified, characterized, and compared the biochemical activities of three M. xanthus RtcB paralogs. We found that M. xanthus RtcB1 resembles E. coli RtcB in its ability to perform intra-and intermolecular sealing of a HO RNAp substrate and capping of a DNA 3=-PO 4 end. M. xanthus RtcB2 can splice HO RNAp but has 5-fold-lower RNA ligase specific activity than RtcB1. In contrast, M. xanthus RtcB3 is distinctively feeble at ligating the HO RNAp substrate, although it readily caps a DNA 3=-PO 4 end. The novelty of M. xanthus RtcB3 is its capacity to cap DNA and RNA 5=-PO 4 ends to form GppDNA and GppRNA products, respectively. As such, RtcB3 joins a growing list of enzymes (including RNA 3=-phosphate cyclase RtcA and thermophilic ATP-dependent RNA ligases) that can cap either end of a polynucleotide substrate. GppDNA formed by RtcB3 can be decapped to pDNA by the DNA repair enzyme aprataxin. IMPORTANCE RtcB enzymes comprise a widely distributed family of RNA 3=-PO 4 ligases distinguished by their formation of 3=-GMP-cappedRNAppG and/or DNAppG polynucleotides. The mechanism and biochemical repertoire of E. coli RtcB are well studied, but it is unclear whether its properties apply to the many bacteria that have genes encoding multiple RtcB paralogs. A comparison of the biochemical activities of three M. xanthus paralogs, RtcB1, RtcB2, and RtcB3, shows that not all RtcBs are created equal. The standout findings concern RtcB3, which is (i) inactive as an RNA 3=-PO 4 ligase but adept at capping a DNA 3=-PO 4 end and (ii) able to cap DNA and RNA 5=-PO 4 ends to form GppDNA and GppRNA, respectively. The GppDNA and GppRNA capping reactions are novel nucleic acid modifications. 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 2ϩ as a cofactor (5-7). RtcB first reacts with GTP to form a covalent RtcB-(histidinyl-N)-GMP intermediate. It then hydrolyzes the RNA 2=,3=-cyclic-PO 4 end to a 3=-PO 4 end and transfers guanylate to the RNA 3=-PO 4 end 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 canon of nucleic acid enzymology, which held that the synthesis of polynucleotide 3=-5= phosphodiesters proceeds via the ...

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Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases

Cited by 13 publications

References 34 publications

Structural and mutational analysis of archaeal ATP-dependent RNA ligase identifies amino acids required for RNA binding and catalysis

Structural and mutational analysis of archaeal ATP-dependent RNA ligase identifies amino acids required for RNA binding and catalysis

DNA_3′pp_5′G de-capping activity of aprataxin: effect of cap nucleoside analogs and structural basis for guanosine recognition

Characterization of 3′-Phosphate RNA Ligase Paralogs RtcB1, RtcB2, and RtcB3 from Myxococcus xanthus Highlights DNA and RNA 5′-Phosphate Capping Activity of RtcB3

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Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases

Cited by 13 publications

References 34 publications

Structural and mutational analysis of archaeal ATP-dependent RNA ligase identifies amino acids required for RNA binding and catalysis

Structural and mutational analysis of archaeal ATP-dependent RNA ligase identifies amino acids required for RNA binding and catalysis

DNA3′pp5′G de-capping activity of aprataxin: effect of cap nucleoside analogs and structural basis for guanosine recognition

Characterization of 3′-Phosphate RNA Ligase Paralogs RtcB1, RtcB2, and RtcB3 from Myxococcus xanthus Highlights DNA and RNA 5′-Phosphate Capping Activity of RtcB3

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DNA_3′pp_5′G de-capping activity of aprataxin: effect of cap nucleoside analogs and structural basis for guanosine recognition