Identification of residues in the Mu transposase essential for catalysis.

Baker, Tania A.; Luo, Li

doi:10.1073/pnas.91.14.6654

Cited by 125 publications

(88 citation statements)

References 50 publications

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“…The essential acidic amino acids have been sug gested to form part of the active site for both cleavage and strand transfer by coordinating divalent cations (Mg^"^ or Mn^-^) (Engelman and Craigie 1992;Kulkosky et al 1992;Bushman et al 1993). Some activity is re stored to the D269N and E392Q MuA derivatives by ad dition of high concentrations of Mn^ '^ in support of this suggestion (Baker and Luo 1994).…”

supporting

confidence: 58%

“…Retroviral inte grases have three highly conserved acidic amino acids termed the D-D-35-E motif, as the second aspartate (D) and the glutamate (E) are separated by 35 residues; these residues of the HIV and Rous sarcoma virus (RSV) inte grases are important for both cleavage and strand transfer in vitro (Drelich et al 1992;Engelman and Craigie 1992;Kulkosky et al 1992;Lefemina et al 1992;van Gent et al 1992;Leavitt et al 1993). We have recently identified acidic residues in MuA that are required for both DNA cleavage and strand transfer (Baker and Luo 1994). The similar effect of mutations, as well as amino acid se-Mu tiansposition lequires four active monomers quence similarity, argue that the essential acidic amino acids of MuA (D269 and E392) are analogs of those of the retroviral integrases (Baker and Luo 1994).…”

mentioning

confidence: 99%

“…We have recently identified acidic residues in MuA that are required for both DNA cleavage and strand transfer (Baker and Luo 1994). The similar effect of mutations, as well as amino acid se-Mu tiansposition lequires four active monomers quence similarity, argue that the essential acidic amino acids of MuA (D269 and E392) are analogs of those of the retroviral integrases (Baker and Luo 1994). Convincing D-D-35-E motifs have been detected in many transposi tion proteins, including those from Tn7, Tn552, IS3, and mariner elements (Fayet et al 1990;Rowland and Dyke 1990;Robertson 1993;Baker and Luo 1994;Radstrom et al 1994) and a related motif has been identified in the proteins encoded by excising elements from ciliated pro tozoa (Doak et al 1994).…”

mentioning

confidence: 99%

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Complete transposition requires four active monomers in the mu transposase tetramer.

Baker¹,

Kremenstova²,

Luo³

1994

Genes Dev.

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A tetramer of Mu transposase (MuA) cleaves and joins multiple DNA strands to promote transposition. Derivatives of MuA altered at two acidic residues that are conserved among transposases and retroviral integrases form tetramers but are defective in both cleavage and joining. These mutant proteins were used to analyze the contribution of individual monomers to the activity of the tetramer. The performance of different protein combinations demonstrates that not all monomers need to be catalytically competent for the complex to promote an individual cleavage or joining reaction. Furthermore, the results indicate that each pair of essential residues is probably donated to the active complex by a single monomer. Although stable, tetramers composed of a mixture of mutant and wild-type MuA generate products cleaved at only one end and with only one end joined to the target DNA. The abundance of these abortive products and the ratios of the two proteins in complexes stalled at different steps indicate that the complete reaction requires the activity of all four monomers. Thus, each subunit of MuA appears to use the conserved acidic amino acids to promote one DNA cleavage or one DNA joining reaction.

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supporting

confidence: 58%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Complete transposition requires four active monomers in the mu transposase tetramer.

Baker¹,

Kremenstova²,

Luo³

1994

Genes Dev.

Self Cite

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“…MuA was purified as described by Baker et al (45). HU was purified as described by Baker and Luo (11). Wild-type MuB and MuBins101N were purified as described by Yamauchi and Baker (20).…”

Section: Dnamentioning

confidence: 99%

“…Transposition is mediated by two Mu-encoded proteins: MuA and MuB. MuA, the transposase, is a member of the transposase/retroviral integrase protein superfamily (11,12). MuA binds specific sites at each end of the Mu genome (13).…”

mentioning

confidence: 99%

Dissecting the roles of MuB in Mu transposition: ATP regulation of DNA binding is not essential for target delivery

Schweidenback¹,

Baker

2008

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

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Regions of RAG1 protein critical for V(D)J recombination

1996

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The products of the recombination activating genes RAG1 and RAG2 are essential for activating V(D)J recombination, and thus are indispensable for the production of functional and diverse antigen receptors. To investigate the function of RAG1, we have tested a series of insertion and substitution mutation for their ability to induce V(D)J rearrangement on both deletional and inversional plasmid substrates. With these substrates we were also able to assess the effects of these mutations on both coding and signal joint formation, and to show that any one mutant affected all these reactions similarly. As defined previously, the core active regions of RAG1 and RAG2 permit the deletion of 40% and 25%, respectively, of well-conversed sequence. We show here that this "dispensable" region of RAG1 is not necessary for coding joint formation or recombination of an integrated substrate, and this portion is not functionally redundant with the "dispensable" region of RAG2. Recombination with these core regions is also still subject to the 12/23 joining rule. Further, the minimal essential core region of RAG1 can be located within an even smaller portion of the gene.

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Identification of residues in the Mu transposase essential for catalysis.

Cited by 125 publications

References 50 publications

Complete transposition requires four active monomers in the mu transposase tetramer.

Complete transposition requires four active monomers in the mu transposase tetramer.

Dissecting the roles of MuB in Mu transposition: ATP regulation of DNA binding is not essential for target delivery

Regions of RAG1 protein critical for V(D)J recombination

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