Dissecting the Chemistry of Protein Splicing and Its Applications

Noren, Christopher J.; Wang, Jimin; Perler, Francine B.

doi:10.1002/(sici)1521-3773(20000204)39:3<450::aid-anie450>3.3.co;2-6

Cited by 86 publications

(126 citation statements)

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“…The currently accepted mechanism for protein splicing could be considered a solution-phase counterpart to this mechanism in which the hydroxyl oxygen of the Ser side chain attacks the carbonyl carbon to form a five-membered intermediate. 41 The presence of His in a peptide sequence has also been reported to enhance the neutral loss of water from sequence ions, perhaps through a neighboring group pathway involving induction of water loss by the nucleophilic side chain on the adjacent protonated carbonyl. 36 The presence of His in peptides could promote the neutral loss of water (as seen in Figure 10).…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

Statistical Characterization of Ion Trap Tandem Mass Spectra from Doubly Charged Tryptic Peptides

et al. 2003

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Collision-induced dissociation (CID) is a common ion activation technique used to energize massselected peptide ions during tandem mass spectrometry. Characteristic fragment ions form from the cleavage of amide bonds within a peptide undergoing CID, allowing the inference of its amino acid sequence. The statistical characterization of these fragment ions is essential for improving peptide identification algorithms and for understanding the complex reactions taking place during CID. An examination of 1465 ion trap spectra from doubly charged tryptic peptides reveals several trends important to understanding this fragmentation process. While less abundant than y ions, b ions are present in sufficient numbers to aid sequencing algorithms. Fragment ions exhibit a characteristic series-specific relationship between their masses and intensities. Each residue influences fragmentation at adjacent amide bonds, with Pro quantifiably enhancing cleavage at its N-terminal amide bond and His increasing the formation of b ions at its C-terminal amide bond. Fragment ions corresponding to a formal loss of ammonia appear preferentially in peptides containing Gln and Asn. These trends are partially responsible for the complexity of peptide tandem mass spectra.Tandem mass spectrometry (MS/MS) of peptides is a central technology for proteomics, enabling the identification of thousands of peptides from a complex mixture. [1][2][3][4] This increasingly widespread technique relies upon the fragmentation of peptides by collisioninduced dissociation (CID), but the chemistry behind the fragmentation process is complex and not comprehensively understood. [5][6][7][8] Peptides undergo CID after they are isolated from other ions by their mass-to-charge (m/z) ratios. Peptides in an acidic solution are introduced to the vacuum of the mass spectrometer via electrospray ionization. 9 The peptide ions are accelerated during CID, leading to more energetic collisions with the ion trap's inert gas molecules. The mobile proton model 10 describes how the added internal energy causes the ionizing proton(s) on each peptide to transfer intramolecularly until one destabilizes a peptide bond, resulting in the cleavage of that bond and the production of two fragments. While more energetic techniques may cleave many classes of bonds within the peptide structure, low-energy CID preferentially breaks the amide bonds. Once the fragment ions are produced, the mass spectrometer records their m/z ratios in a tandem mass spectrum.Determining the sequence of a peptide from its tandem spectrum is complicated by the variety and variability of the fragment ions produced. Cleavage of amide bonds results in b and y Figure 1). b ions may fragment further to produce a ions. 13 If only these three ions were produced for every amide bond in a 10-residue peptide, the fragment ion spectrum would contain 27 peaks. This ideal spectrum differs from experimental spectra as a result of several causes. First, a subset of the expected fragment ions may not be present. Second...

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Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

Statistical Characterization of Ion Trap Tandem Mass Spectra from Doubly Charged Tryptic Peptides

et al. 2003

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“…[12][13][14][15][16]. The hallmark of homing endonucleases, like restriction enzymes, is their ability to cleave double-stranded DNA at specific target sites (12,17).…”

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“…The catalytic centers carry two essential aspartate residues in the LAGLIDADG motif. These residues function by coordinating a divalent cation necessary for catalysis, Mg 2ϩ being the preferred metal ion (12)(13)(14)(15)(16).…”

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confidence: 99%

Mycobacterium tuberculosis RecA Intein Possesses a Novel ATP-dependent Site-specific Double-stranded DNA Endonuclease Activity

Guhan

Muniyappa

2002

Journal of Biological Chemistry

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Mycobacterium tuberculosis recA harbors an intervening sequence in its open reading frame, presumed to encode an endonuclease (PI-MtuI) required for intein homing in inteinless recA allele. Although the proteinsplicing ability of PI-MtuI has been characterized, the identification of its putative endonuclease activity has remained elusive. To investigate whether PI-MtuI possesses endonuclease activity, recA intervening sequence was cloned, overexpressed, and purified to homogeneity. Here we show that PI-MtuI bound both single-and double-stranded DNA with similar affinity but failed to cleave DNA in the absence of cofactors. Significantly, PI-MtuI nicked supercoiled DNA in the presence of alternative cofactors but required both Mn 2؉ and ATP to generate linear double-stranded DNA. We observed that PI-MtuI was able to inflict a staggered double-strand break 24 bp upstream of the insertion site in the inteinless recA allele. Similar to a few homing endonucleases, DNA cleavage by PI-MtuI was specific with an exceptionally long cleavage site spanning 22 bp. The kinetic mechanism of PI-MtuI promoted cleavage supports a sequential rather than concerted pathway of strand cleavage with the formation of nicked double-stranded DNA as an intermediate. Together, these results reveal that RecA intein is a novel Mn 2؉ -ATP-dependent doublestrand specific endonuclease, which is likely to be important for homing process in vivo.The prototype Escherichia coli RecA protein plays a central role in homologous recombination, DNA repair, restoration of stalled replication forks and SOS response (reviewed in Refs.

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“…The genes for homing endonucleases are found among group I and group II introns, archaeal introns, intein-coding sequences, and free standing open reading frames (reviewed in Refs. [1][2][3][4][5][6]. Inteins are genetic elements present within protein-coding sequences with dual function: protein-splicing and homing endonuclease activities.…”

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confidence: 99%

The RecA Intein of Mycobacterium tuberculosisPromotes Cleavage of Ectopic DNA Sites

Guhan

Muniyappa

2002

Journal of Biological Chemistry

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The RecA intein of Mycobacterium tuberculosis, a novel double-stranded DNA endonuclease, requires both Mn 2؉ and ATP for efficient cleavage of the inteinless recA allele. In this study, we show that Mg 2؉ alone was sufficient to stimulate PI-MtuI to cleave doublestranded DNA at ectopic sites. In the absence of Mg 2؉ , PI-MtuI formed complexes with topologically different forms of DNA containing ectopic recognition sequences with equal affinity but failed to cleave DNA. We observed that PI-MtuI was able to inflict double-strand breaks robustly within the ectopic recognition sequence to generate either a blunt end or 1-2-nucleotide 3-hydroxyl overhangs. Mobile inteins and introns are genetic elements capable of self-propagation by "homing" into host genes in a wide variety of organisms: eubacteria, eukarya, archaea, and viruses (reviewed in Ref. 1). The process is promoted by a homing endonuclease, which is encoded by an open reading frame embedded within the genetic element. The genes for homing endonucleases are found among group I and group II introns, archaeal introns, intein-coding sequences, and free standing open reading frames (reviewed in Refs. 1-6). Inteins are genetic elements present within protein-coding sequences with dual function: protein-splicing and homing endonuclease activities. They are believed to play a central role in rearrangement of organelle as well as nuclear genomes (1-6). The hallmark of homing endonucleases is their ability to recognize and cleave extended asymmetric sequences (14 -40 bp) that are generally centered on the intein insertion site in inteinless alleles (1, 7). Homing endonucleases can be classified into four families based on the presence of conserved motifs: LAGLIDADG, GIY-YIG, His-Cys box, and H-N-H (1,5,6,8). Among these, the LAGLIDADG family is the largest, widespread and much studied class of homing endonucleases. Structural and biochemical studies have demonstrated that homing endonucleases with one LAGLIDADG motif act as homodimers, whereas enzymes with two such motifs function as monomers during catalysis (9 -12). Under standard assay conditions in vitro, these enzymes are extremely specific for their recognition sites. However, recent evidence suggests that self-splicing group II introns transpose into ectopic DNA sites that resemble their natural homing sites (13,14). The transposition of group II introns involves reverse splicing of the intron into the intronless allele, which are then reverse transcribed to give complementary DNA. Following the synthesis of the second strand, the intron is incorporated into the genomic DNA by homologous recombination (1-6). But equivalent information is not available for any intein endonuclease. The exact sequence of events that lead to recognition and cleavage of DNA by homing endonucleases is poorly understood. In addition, in no case the molecular mechanism underlying cleavage of ectopic DNA sites by an intein endonuclease has been elucidated.Mycobacterium tuberculosis RecA intein (PI-MtuI) 1 is a member of the LAGLIDADG...

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Dissecting the Chemistry of Protein Splicing and Its Applications

Cited by 86 publications

References 0 publications

Statistical Characterization of Ion Trap Tandem Mass Spectra from Doubly Charged Tryptic Peptides

Statistical Characterization of Ion Trap Tandem Mass Spectra from Doubly Charged Tryptic Peptides

Mycobacterium tuberculosis RecA Intein Possesses a Novel ATP-dependent Site-specific Double-stranded DNA Endonuclease Activity

The RecA Intein of Mycobacterium tuberculosisPromotes Cleavage of Ectopic DNA Sites

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