Structural Domains in the Type III Restriction Endonuclease EcoP15I: Characterization by Limited Proteolysis, Mass Spectrometry and Insertional Mutagenesis

Wagenführ, Katja; Pieper, Stefan; Mackeldanz, Petra; Linscheid, Michael; Krüger, Detlev H.; Reuter, Monika

doi:10.1016/j.jmb.2006.10.087

Cited by 11 publications

(9 citation statements)

References 21 publications

(32 reference statements)

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“…These sites can be several thousands of base pairs apart and under standard conditions cleavage occurs 24–28 bp in 3′-direction from one of them (3,5,6). However, very conflicting data exist about the movement of EcoP15I between remote DNA sites.…”

Section: Introductionmentioning

confidence: 99%

“…Results obtained from limited proteolysis experiments and mass spectrometry showed that the Res subunit of EcoP15I is comprised of two stable domains connected by a flexible linker (5). According to these experiments, the N-terminal part (amino acids 40–705) of EcoP15I Res subunit appears to be the motor or translocase domain (Tr) containing conserved helicase motifs, whereas the C-terminal part (amino acids 729–966) is the endonuclease domain (E) carrying the active site responsible for DNA cleavage.…”

Section: Introductionmentioning

confidence: 99%

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Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several DNA-binding regions and ATPase activity

Wyszomirski

Curth

Alves

et al. 2011

Nucleic Acids Research

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For efficient DNA cleavage, the Type III restriction endonuclease EcoP15I communicates with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of methylation (Mod) and restriction (Res) subunits forming a multifunctional enzyme complex able to methylate or to cleave DNA. In this study, we determined by different analytical methods that EcoP15I contains a single Res subunit in a Mod2Res stoichiometry. The Res subunit comprises a translocase (Tr) domain carrying functional motifs of superfamily 2 helicases and an endonuclease domain with a PD..D/EXK motif. We show that the isolated Tr domain retains ATP-hydrolyzing activity and binds single- and double-stranded DNA in a sequence-independent manner. To localize the regions of DNA binding, we screened peptide arrays representing the entire Res sequence for their ability to interact with DNA. We discovered four DNA-binding regions in the Tr domain and two DNA-binding regions in the endonuclease domain. Modelling of the Tr domain shows that these multiple DNA-binding regions are located on the surface, free to interact with DNA. Interestingly, the positions of the DNA-binding regions are conserved among other Type III restriction endonucleases.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several DNA-binding regions and ATPase activity

Wyszomirski

Curth

Alves

et al. 2011

Nucleic Acids Research

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“…[3][4][5] It was consistently seen that using an equimolar ratio of EcoP15I proteins to recognition sites resulted in DNA cleavage occurring at only one of the interacting sites where it cuts with considerable imprecision: 24 to 26 bases downstream of 5′-CAGCAG and 26 to 28 bases upstream of 5′-CTGCTG. [6][7][8][9] However, divergent opinions about how the long-distance interaction between the recognition sites is realized currently exist. Some models favour ATP-driven DNA translocation alone 3 or combined with three-dimensional DNA looping, 10,11 and corresponding loop structures have been visualized by atomic force microscopy and fast-scan atomic force microscopy.…”

Section: Introductionmentioning

confidence: 99%

Functional Characterization and Modulation of the DNA Cleavage Efficiency of Type III Restriction Endonuclease EcoP15I in Its Interaction with Two Sites in the DNA Target

Möncke‐Buchner

Rothenberg

Reich

et al. 2009

Journal of Molecular Biology

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“…Mt BPL exhibit restricted cleavage in the presence of substrate suggesting that scissile site interacts with the substrate. While the biotin binding site is constituted by the conserved ‘GRGRHGR’ in both the BPLs but binding of biotin/bio-5′AMP promotes conformational change in Ec BirA [28], [29].…”

Section: Discussionmentioning

confidence: 99%

Diversity in Functional Organization of Class I and Class II Biotin Protein Ligase

et al. 2011

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The cell envelope of Mycobacterium tuberculosis (M.tuberculosis) is composed of a variety of lipids including mycolic acids, sulpholipids, lipoarabinomannans, etc., which impart rigidity crucial for its survival and pathogenesis. Acyl CoA carboxylase (ACC) provides malonyl-CoA and methylmalonyl-CoA, committed precursors for fatty acid and essential for mycolic acid synthesis respectively. Biotin Protein Ligase (BPL/BirA) activates apo-biotin carboxyl carrier protein (BCCP) by biotinylating it to an active holo-BCCP. A minimal peptide (Schatz), an efficient substrate for Escherichia coli BirA, failed to serve as substrate for M. tuberculosis Biotin Protein Ligase (MtBPL). MtBPL specifically biotinylates homologous BCCP domain, MtBCCP87, but not EcBCCP87. This is a unique feature of MtBPL as EcBirA lacks such a stringent substrate specificity. This feature is also reflected in the lack of self/promiscuous biotinylation by MtBPL. The N-terminus/HTH domain of EcBirA has the self-biotinable lysine residue that is inhibited in the presence of Schatz peptide, a peptide designed to act as a universal acceptor for EcBirA. This suggests that when biotin is limiting, EcBirA preferentially catalyzes, biotinylation of BCCP over self-biotinylation. R118G mutant of EcBirA showed enhanced self and promiscuous biotinylation but its homologue, R69A MtBPL did not exhibit these properties. The catalytic domain of MtBPL was characterized further by limited proteolysis. Holo-MtBPL is protected from proteolysis by biotinyl-5′ AMP, an intermediate of MtBPL catalyzed reaction. In contrast, apo-MtBPL is completely digested by trypsin within 20 min of co-incubation. Substrate selectivity and inability to promote self biotinylation are exquisite features of MtBPL and are a consequence of the unique molecular mechanism of an enzyme adapted for the high turnover of fatty acid biosynthesis.

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Structural Domains in the Type III Restriction Endonuclease EcoP15I: Characterization by Limited Proteolysis, Mass Spectrometry and Insertional Mutagenesis

Cited by 11 publications

References 21 publications

Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several DNA-binding regions and ATPase activity

Type III restriction endonuclease EcoP15I is a heterotrimeric complex containing one Res subunit with several DNA-binding regions and ATPase activity

Functional Characterization and Modulation of the DNA Cleavage Efficiency of Type III Restriction Endonuclease EcoP15I in Its Interaction with Two Sites in the DNA Target

Diversity in Functional Organization of Class I and Class II Biotin Protein Ligase

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