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Literature summary extracted from

  • Sharma, D.P.; Vijayan, R.; Rehman, S.A.A.; Gourinath, S.
    Structural insights into the interaction of helicase and primase in Mycobacterium tuberculosis (2018), Biochem. J., 475, 3493-3509 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.7.7.101 gene dnaG, recombinant expression of His-tagged wild-type and mutant enzymes, and of unlabeled and selenomethionine-labeled MtDnaG-CTDs in Escherichia coli strain BL21(DE3) Mycobacterium tuberculosis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.7.7.101 purified recombinant C-terminal domain of DnaG, mixing of 0.002 ml of 6 mg/ml native MtDnaG-CTD and 5 mg/ml selenomethionine-labeled MtDnaG-CTD with 0.002 ml of reservoir solution containing 20% w/v PEG 3350, 0.2 M magnesium acetate tetrahydrate, and equilibration against 0.5 ml of reservoir solution, for the SeMet-labeled protein a reeservoir solution of reservoir solution of 18% w/v PEG 3350, 0.15 M magnesium acetate, and MOPS, pH 7.6, is used, 4°C, 1-2 weeks, X-ray diffraction structure determination and analysis at 1.57-1.58 A resolution, modeling Mycobacterium tuberculosis

Protein Variants

EC Number Protein Variants Comment Organism
2.7.7.101 F615A site-directed mutagenesis, mutant MtDnaG-CTD F615A Mycobacterium tuberculosis
2.7.7.101 I605A site-directed mutagenesis, mutant MtDnaG-CTD I605A, binding study shows a significant decrease in the binding affinity of MtDnaB-NTD with the Ile605Ala mutant of MtDnaG-CTD compared with native MtDnaG-CTD Mycobacterium tuberculosis

Metals/Ions

EC Number Metals/Ions Comment Organism Structure
2.7.7.101 Mg2+ required Mycobacterium tuberculosis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.7.7.101 35000
-
 the C-terminal domain MtDnaG-CTD is a dimer in solution, gel filtration Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.7.7.101 ssDNA + n NTP Mycobacterium tuberculosis
-
 ssDNA/pppN(pN)n-1 hybrid + (n-1) diphosphate
-
 ?
2.7.7.101 ssDNA + n NTP Mycobacterium tuberculosis H37Rv
-
 ssDNA/pppN(pN)n-1 hybrid + (n-1) diphosphate
-
 ?
2.7.7.101 ssDNA + n NTP Mycobacterium tuberculosis ATCC 25618
-
 ssDNA/pppN(pN)n-1 hybrid + (n-1) diphosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.7.7.101 Mycobacterium tuberculosis P9WNW1
-
-
2.7.7.101 Mycobacterium tuberculosis ATCC 25618 P9WNW1
-
-
2.7.7.101 Mycobacterium tuberculosis H37Rv P9WNW1
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.7.7.101 recombinant His-tagged -type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, ultrafiltration, and gel filtration Mycobacterium tuberculosis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.7.7.101 ssDNA + n NTP
-
 Mycobacterium tuberculosis ssDNA/pppN(pN)n-1 hybrid + (n-1) diphosphate
-
 ?
2.7.7.101 ssDNA + n NTP
-
 Mycobacterium tuberculosis H37Rv ssDNA/pppN(pN)n-1 hybrid + (n-1) diphosphate
-
 ?
2.7.7.101 ssDNA + n NTP
-
 Mycobacterium tuberculosis ATCC 25618 ssDNA/pppN(pN)n-1 hybrid + (n-1) diphosphate
-
 ?

Subunits

EC Number Subunits Comment Organism
2.7.7.101 More MtDnaG-CTD interacts at the dimer-dimer interface of MtDnaB-NTD using mostly hydrophobic residues of the helical hairpin regions (HHRs). The dimer-dimer interface of MtDnaB-NTD is considered as a DnaG-binding site Mycobacterium tuberculosis

Synonyms

EC Number Synonyms Comment Organism
2.7.7.101 DnaG
-
 Mycobacterium tuberculosis

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.7.7.101 37
-
 assay at Mycobacterium tuberculosis

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.7.7.101 7.5
-
 assay at Mycobacterium tuberculosis

General Information

EC Number General Information Comment Organism
2.7.7.101 evolution functional conservation despite the low sequence homology of the DnaB-binding domains of DnaGs of eubacteria. MtDnaB-NTD showed micromolar affinity with DnaG-CTDs from Escherichia coli and Helicobacter pylori and unstable binding with DnaG-CTD from Vibrio cholerae. The interacting domains of both DnaG and DnaB demonstrate the species-specific evolution of the replication initiation system Mycobacterium tuberculosis
2.7.7.101 additional information structure modeling using the MtDnaG-CTD and MtDnaB-NTD crystal structures for analysis of crucial helicase-primase interaction in Mycobacterium tuberculosis. Two nonconserved hydrophobic residues (Ile605 and Phe615) of MtDnaG are identified as potential key residues interacting with MtDnaB. The loop, connecting the two helices of the HHR, is concluded to be largely responsible for the stability of the DnaB-DnaG complex. Molecular dynamic simulations were performed on the models of the MtDnaB-NTD complex with DnaG-CTD, DnaG-CTD I605A, and DnaG-CTD F615A. Structure comparisons of the enzyme from Mycobacterium tuberculosis with enzymes from other species. Residue Ile605 in the HHR is crucial for helicase-primase interaction. The dimer-dimer interface of MtDnaB-NTD is considered as a DnaG-binding site. Mycobacterium DnaB-NTD interacts with DnaG-CTD of other organisms with low binding affinity Mycobacterium tuberculosis
2.7.7.101 physiological function the helicase-primase interaction is an essential event in DNA replication and is mediated by the highly variable C-terminal domain (CTD) of primase (DnaG) and N-terminal domain (NTD) of helicase (DnaB) Mycobacterium tuberculosis