Crystal structures of Escherichia coli exonuclease I in complex with single-stranded DNA provide insights into the mechanism of processive digestion

Korada, Sai Krishna C.; Johns, Trevor D.; Smith, Christopher E.; Jones, Nathan D.; McCabe, Kimberly A.; Bell, Charles E.

doi:10.1093/nar/gkt278

Cited by 21 publications

(33 citation statements)

References 34 publications

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“…Most notably, residues S 108 , R 130 , S 135 and the hydroxyl group on Y 129 (S 111 , R 133 , S 138 , and Y 132 in REXO2) form hydrogen bonds with the 5’ phosphate of pGpG, capping the substrate. This phosphate cap creates a major constriction of the active site, which is not observed in structurally related 3’-5’ exoribonucleases such as RNase T or ExoI (Figures 2C, S1C and S1D) 32–34 . RNase T and ExoI accommodate longer RNA substrates, facilitated by an expansive active site.…”

Section: Resultsmentioning

confidence: 89%

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Oligoribonuclease functions as a diribonucleotidase to bypass a key bottleneck in RNA degradation

et al. 2019

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23Degradation of RNA polymers is a multistep process catalyzed by specific subsets of RNases. In 24 all cases, degradation is completed by exoribonucleases that recycle RNA fragments into 25 nucleotide monophosphate. In γ-proteobacteria, a group of up to eight 3'-5' exoribonucleases 26 have been implicated in RNA degradation. Oligoribonuclease (Orn) is unique among them as its 27 activity is required for clearing short RNA fragments, a function important for cellular fitness. 28 However, the mechanistic basis for this substrate selectivity remained unclear. Here we show 29 that Orn's activity as a general exoribonuclease has been vastly overestimated by demonstrating 30 that the enzyme exhibits a much narrower substrate preference for diribonucleotides. Co-crystal 31 structures of Orn with substrates reveal an active site optimized for diribonucleotides that does 32 not accommodate longer substrates. While other cellular RNases process oligoribonucleotides 33 down to diribonucleotide entities, our functional studies demonstrate that Orn is the one and 34 only diribonucleotidase that completes the final stage of the RNA degradation pathway. 35Together, these results indicate that Orn is a dedicated diribonucleotidase that clears the 36 diribonucleotide pool that otherwise affects cellular physiology and viability. 37 38 14 . Since the discovery of c-di-GMP over 30 years ago, it has been known that the signal is degraded 56 by a two-step process with a linear pGpG diribonucleotide intermediate 15 . While the enzyme for 57 linearizing c-di-GMP to pGpG was discovered early on 16,17 , the identity of the enzyme that degrades 58 pGpG remained elusive. Two recent studies showed that Orn is the primary enzyme that degrades 59 pGpG in Pseudomonas aeruginosa 18,19 . In an orn deletion strain, the accrual of linear pGpG has a 60 profound effect on cells. Specifically, the increase in pGpG inhibits upstream phosphodiesterases that 61 degrade c-di-GMP, thereby triggering phenotypes associated with high cellular c-di-GMP levels. 62 However, the molecular basis of Orn's unique cellular functions in γ-proteobacteria that distinguishes 63 it from all other exoribonucleases remains unexplained. 64Since its discovery over 50 years ago 20 , Orn has been presumed to degrade 65 oligoribonucleotides 21-23 . This notion largely derived from assays utilizing two types of substrates. In 66 one series of experiments, 3 H polyuridine (poly(U)) was incubated with Orn, other enzymes, or lysates 67 and analyzed by paper chromatography, which offers limited resolution overall [21][22][23] . In a second set of 68 experiments, Orn was incubated with oligoribonucleotides that had been tagged at their 5' terminus by 69

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Section: Resultsmentioning

confidence: 89%

“…(C) Active-site view of E. coli RNase T bound to dT13 (PDB 3v9z; 32 ). (D) Active-site view of E. coli Exo I bound to dAAC (PDB 4jrp; 34 ).…”

Section: Figure S1mentioning

confidence: 99%

Oligoribonuclease functions as a diribonucleotidase to bypass a key bottleneck in RNA degradation

et al. 2019

Preprint

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“…As examples, CRN-4 and NP exonuclease prefer to digest double-stranded DNA or RNA. 3,4 Exo I and ISG20 prefer to digest single-stranded DNA or RNA, 5,6 while Snp, 3′hExo, and RNase T digest 3′ overhang of duplex nucleic acids. 7,8 DEDDh exonucleases play essential roles in various cellular processes, including RNA maturation, RNA turnover, DNA replication, and DNA repair.…”

Section: ■ Introductionmentioning

confidence: 99%

Identification of Inhibitors for the DEDDh Family of Exonucleases and a Unique Inhibition Mechanism by Crystal Structure Analysis of CRN-4 Bound with 2-Morpholin-4-ylethanesulfonate (MES)

et al. 2016

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The DEDDh family of exonucleases plays essential roles in DNA and RNA metabolism in all kingdoms of life. Several viral and human DEDDh exonucleases can serve as antiviral drug targets due to their critical roles in virus replication. Here using RNase T and CRN-4 as the model systems, we identify potential inhibitors for DEDDh exonucleases. We further show that two of the inhibitors, ATA and PV6R, indeed inhibit the exonuclease activity of the viral protein NP exonuclease of Lassa fever virus in vitro. Moreover, we determine the crystal structure of CRN-4 in complex with MES that reveals a unique inhibition mechanism by inducing the general base His179 to shift out of the active site. Our results not only provide the structural basis for the inhibition mechanism but also suggest potential lead inhibitors for the DEDDh exonucleases that may pave the way for designing nuclease inhibitors for biochemical and biomedical applications.

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“…Extensive research has focused on elucidating the mechanism for its high processivity [ 5 , 6 ]. Recently, the crystal structure of Exo I complexed with ssDNA revealed the topological linkage and two-site substrate binding underlying the high processivity [ 6 ].…”

Section: Introductionmentioning

confidence: 99%

“…Both Exo I and the exonuclease domain of DNA polymerase I belong to the DnaQ superfamily (or DEDD superfamily) based on sequence analysis [ 5 , 11 , 12 ]. According to the two-metal ion mechanism, two magnesium ions are essential for exonuclease activity, with Mg B binding directly to the enzyme and Mg A binding via the 3' end of the bound ssDNA substrate [ 6 , 9 ]. The contribution of two metal ions to thermal stability of Exo I has not been characterized.…”

Section: Introductionmentioning

confidence: 99%

Structural and Biochemical Studies of a Moderately Thermophilic Exonuclease I from Methylocaldum szegediense

et al. 2015

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A novel exonuclease, designated as MszExo I, was cloned from Methylocaldum szegediense, a moderately thermophilic methanotroph. It specifically digests single-stranded DNA in the 3ʹ to 5ʹ direction. The protein is composed of 479 amino acids, and it shares 47% sequence identity with E. coli Exo I. The crystal structure of MszExo I was determined to a resolution of 2.2 Å and it aligns well with that of E. coli Exo I. Comparative studies revealed that MszExo I and E. coli Exo I have similar metal ion binding affinity and similar activity at mesophilic temperatures (25–47°C). However, the optimum working temperature of MszExo I is 10°C higher, and the melting temperature is more than 4°C higher as evaluated by both thermal inactivation assays and DSC measurements. More importantly, two thermal transitions during unfolding of MszExo I were monitored by DSC while only one transition was found in E. coli Exo I. Further analyses showed that magnesium ions not only confer structural stability, but also affect the unfolding of MszExo I. MszExo I is the first reported enzyme in the DNA repair systems of moderately thermophilic bacteria, which are predicted to have more efficient DNA repair systems than mesophilic ones.

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Crystal structures of Escherichia coli exonuclease I in complex with single-stranded DNA provide insights into the mechanism of processive digestion

Cited by 21 publications

References 34 publications

Oligoribonuclease functions as a diribonucleotidase to bypass a key bottleneck in RNA degradation

Oligoribonuclease functions as a diribonucleotidase to bypass a key bottleneck in RNA degradation

Identification of Inhibitors for the DEDDh Family of Exonucleases and a Unique Inhibition Mechanism by Crystal Structure Analysis of CRN-4 Bound with 2-Morpholin-4-ylethanesulfonate (MES)

Structural and Biochemical Studies of a Moderately Thermophilic Exonuclease I from Methylocaldum szegediense

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