The helicase–primase interaction is an essential event in DNA replication and is mediated by the highly variable C-terminal domain of primase (DnaG) and N-terminal domain of helicase (DnaB). To understand the functional conservation despite the low sequence homology of the DnaB-binding domains of DnaGs of eubacteria, we determined the crystal structure of the helicase-binding domain of DnaG from Mycobacterium tuberculosis (MtDnaG-CTD) and did so to a resolution of 1.58 Å. We observed the overall structure of MtDnaG-CTD to consist of two subdomains, the N-terminal globular region (GR) and the C-terminal helical hairpin region (HHR), connected by a small loop. Despite differences in some of its helices, the globular region was found to have broadly similar arrangements across the species, whereas the helical hairpins showed different orientations. To gain insights into the crucial helicase–primase interaction in M. tuberculosis, a complex was modeled using the MtDnaG-CTD and MtDnaB-NTD crystal structures. Two nonconserved hydrophobic residues (Ile605 and Phe615) of MtDnaG were identified as potential key residues interacting with MtDnaB. Biosensor-binding studies showed a significant decrease in the binding affinity of MtDnaB-NTD with the Ile605Ala mutant of MtDnaG-CTD compared with native MtDnaG-CTD. The loop, connecting the two helices of the HHR, was concluded to be largely responsible for the stability of the DnaB–DnaG complex. Also, 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.
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Cover Image
Cover Image
The cover image shows an artistic representation of the fluorescence-based assay of deadenylase activity used by Pavanello et al. in this issue, with fluorescence proportional to enzyme activity. Fluorescence (λem = 528 nm) was detected in a multi-mode reader using 96-well plates. Activity of different enzyme complexes (left to right) was measured at various time points (top to bottom). Controls (inactive enzyme preparations and no enzyme controls) were also included (bottom two rows). Pavanello et al. report that the central region of CNOT1 and CNOT9 stimulates deadenylation by the Ccr4–Not nuclease module (see pages 3437–3450).
Structural insights into the interaction of helicase and primase in Mycobacterium tuberculosis
Dhakaram Pangeni Sharma, Ramachandran Vijayan, Syed Arif Abdul Rehman, Samudrala Gourinath; Structural insights into the interaction of helicase and primase in Mycobacterium tuberculosis. Biochem J 15 November 2018; 475 (21): 3493–3509. doi: https://doi.org/10.1042/BCJ20180673
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