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Papers by Kiran Chintakayala

Nucleic Acids Research, 2015

Curved DNA binding protein A (CbpA) is a cochaperone and nucleoid associated DNA binding protein ... more Curved DNA binding protein A (CbpA) is a cochaperone and nucleoid associated DNA binding protein conserved in most ␥-proteobacteria. Best studied in Escherichia coli, CbpA accumulates to >2500 copies per cell during periods of starvation and forms aggregates with DNA. However, the molecular basis for DNA binding is unknown; CbpA lacks motifs found in other bacterial DNA binding proteins. Here, we have used a combination of genetics and biochemistry to elucidate the mechanism of DNA recognition by CbpA. We show that CbpA interacts with the DNA minor groove. This interaction requires a highly conserved arginine side chain. Substitution of this residue, R116, with alanine, specifically disrupts DNA binding by CbpA, and its homologues from other bacteria, whilst not affecting other CbpA activities. The intracellular distribution of CbpA alters dramatically when DNA binding is negated. Hence, we provide a direct link between DNA binding and the behaviour of CbpA in cells.

PLoS Genetics, 2013

The Escherichia coli curved DNA binding protein A (CbpA) is a poorly characterised nucleoid assoc... more The Escherichia coli curved DNA binding protein A (CbpA) is a poorly characterised nucleoid associated factor and cochaperone. It is expressed at high levels as cells enter stationary phase. Using genetics, biochemistry, and genomics, we have examined regulation of, and DNA binding by, CbpA. We show that Fis, the dominant growth-phase nucleoid protein, prevents CbpA expression in growing cells. Regulation by Fis involves an unusual ''insulation'' mechanism. Thus, Fis protects cbpA from the effects of a distal promoter, located in an adjacent gene. In stationary phase, when Fis levels are low, CbpA binds the E. coli chromosome with a preference for the intrinsically curved Ter macrodomain. Disruption of the cbpA gene prompts dramatic changes in DNA topology. Thus, our work identifies a novel role for Fis and incorporates CbpA into the growing network of factors that mediate bacterial chromosome structure.

Nucleic Acids Research, 2010

Bacterial primase is stimulated by replicative helicase to produce RNA primers that are essential... more Bacterial primase is stimulated by replicative helicase to produce RNA primers that are essential for DNA replication. To identify mechanisms regulating primase activity, we characterized primase initiation specificity and interactions with the replicative helicase for gram-positive Firmicutes (Staphylococcus, Bacillus and Geobacillus) and gram-negative Proteobacteria (Escherichia, Yersinia and Pseudomonas). Contributions of the primase zinc-binding domain, RNA polymerase domain and helicase-binding domain on de novo primer synthesis were determined using mutated, truncated, chimeric and wild-type primases. Key residues in the b4 strand of the primase zinc-binding domain defined class-associated trinucleotide recognition and substitution of these amino acids transferred specificity across classes. A change in template recognition provided functional evidence for interaction in trans between the zinc-binding domain and RNA polymerase domain of two separate primases. Helicase binding to the primase C-terminal helicase-binding domain modulated RNA primer length in a species-specific manner and productive interactions paralleled genetic relatedness. Results demonstrated that primase template specificity is conserved within a bacterial class, whereas the primase-helicase interaction has co-evolved within each species.

Nucleic Acids Research, 2013

The clamp-loader complex plays a crucial role in DNA replication by loading the b-clamp onto prim... more The clamp-loader complex plays a crucial role in DNA replication by loading the b-clamp onto primed DNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry, structure and assembly pathway of this complex, and how it interacts with the replicative helicase, in Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealed that a hetero-pentameric q 3 -d-d 0 Bacillus subtilis clamp-loader assembles via multiple pathways, which differ from those exhibited by the Gram-negative model Escherichia coli. Based on this information, a homology model of the B. subtilis q 3 -d-d 0 complex was constructed, which revealed the spatial positioning of the full C-terminal q domain. The structure of the d subunit was determined by X-ray crystallography and shown to differ from that of E. coli in the nature of the amino acids comprising the q and d 0 binding regions. Most notably, the q-d interaction appears to be hydrophilic in nature compared with the hydrophobic interaction in E. coli. Finally, the interaction between q 3 and the replicative helicase DnaB was driven by ATP/Mg 2+ conformational changes in DnaB, and evidence is provided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilize its interaction with q 3 .

Molecular Microbiology, 2007

The bacterial primase (DnaG)-helicase (DnaB) interaction is mediated by the C-terminal domain of ... more The bacterial primase (DnaG)-helicase (DnaB) interaction is mediated by the C-terminal domain of DnaG (p16) and a linker that joins the N-and C-terminal domains (p17 and p33 respectively) of DnaB. The crystal and nuclear magnetic resonance structures of p16 from Escherichia coli and Bacillus stearothermophilus DnaG proteins revealed a unique structural homology with p17, despite the lack of amino acid sequence similarity. The functional significance of this is not clear. Here, we have employed a 'domain swapping' approach to replace p17 with its structural homologue p16 to create chimeras. p33 alone hydrolyses ATP but exhibits no helicase activity. Fusing p16 (p16-p33) or DnaG (G-p33) to the N-terminus of p33 produced chimeras with partially restored helicase activities. Neither chimera interacted with DnaG. The p16-p33 chimera formed hexamers while G-p33 assembled into tetramers. Furthermore, G-p33 and DnaB formed mixed oligomers with ATPase activity better than that of the DnaB/ DnaG complex and helicase activity better than the sum of the individual DnaB and G-p33 activities but worse than that of the DnaB/DnaG complex. Our com-bined data provide direct evidence that p16 and p17 are not only structural but also functional homologues, albeit their amino acid composition differences are likely to influence their precise roles.

Molecular Microbiology, 2010

The Escherichia coli curved DNA-binding protein A (CbpA) is a nucleoid-associated DNA-binding fac... more The Escherichia coli curved DNA-binding protein A (CbpA) is a nucleoid-associated DNA-binding factor and chaperone that is expressed at high levels as cells enter stationary phase. Using a combination of genetics, biochemistry, structural modelling and singlemolecule atomic force microscopy we have examined dimerization of, and DNA binding by, CbpA. Our data show that CbpA dimerization is driven by a hydrophobic surface comprising amino acid side chains W287 and L290 located on the same side of an a helix close to the C-terminus of CbpA. Derivatives of CbpA that are unable to dimerize are also unable to bind DNA. Free in solution, CbpA can exist as either a monomer or dimer. However, when bound to DNA, CbpA forms large aggregates that can protect DNA from degradation by nucleases. These CbpA-DNA aggregates are similar in morphology to protein-DNA complexes formed by the DNA-binding protein from starved cells (Dps), the only other stationary phase-specific nucleoid protein. Conversely, protein-DNA complexes formed by Fis, the major growth phase nucleoid protein, have a markedly different appearance.

Molecular Microbiology, 2008

The study of primases from model organisms such as Escherichia coli, phage T7 and phage T4 has de... more The study of primases from model organisms such as Escherichia coli, phage T7 and phage T4 has demonstrated the essential nature of primase function, which is to generate de novo RNA polymers to prime DNA polymerase. However, little is known about the function of primases from other eubacteria. Their overall low primary sequence homology may result in functional differences. To help understand which primase functions were conserved, primase and its replication partner helicase from the pathogenic Gram-positive bacteria Staphylococcus aureus were compared in detail with that of E. coli primase and helicase. The conserved properties were to primer initiation and elongation and included slow kinetics, low fidelity and poor sugar specificity. The significant differences included S. aureus primase having sixfold higher kinetic affinity for its template than E. coli primase under equivalent conditions. This naturally higher activity was balanced by its fourfold lower stimulation by its replication fork helicase compared with E. coli primase. The most significant difference between the two primases was that S. aureus helicase stimulation did not broaden the S. aureus primase initiation specificity, which has important biological implications. Fig. 4. Primase concentration dependence of RNA primer synthesis was visualized by denaturing HPLC (left) and quantified (right). The d(CTA) template concentration was 2 mM, the primase concentrations are indicated on the chromatograms and all samples were incubated for 90 min. The dashed line indicates the theoretical stoichiometric case in which one primer would be synthesized per template. The solid line through the quantified data conformed to the quadratic equation for primase-ssDNA complex formation from its components.

Journal of Molecular Biology, 2011

Molecular Microbiology, 2009

During DNA replication the helicase (DnaB) recruits the primase (DnaG) in the replisome to initia... more During DNA replication the helicase (DnaB) recruits the primase (DnaG) in the replisome to initiate the polymerization of new DNA strands. DnaB is attached to the t subunit of the clamp-loader that loads the b clamp and interconnects the core polymerases on the leading and lagging strands. The t-DnaB-DnaG ternary complex is at the heart of the replisome and its function is likely to be modulated by a complex network of allosteric interactions. Using a stable ternary complex comprising the primase and helicase from Geobacillus stearothermophilus and the t subunit of the clamp-loader from Bacillus subtilis we show that changes in the DnaB-t interaction can stimulate allosterically primer synthesis by DnaG in vitro. The A550V t mutant stimulates the primase activity more efficiently than the native protein. Truncation of the last 18 C-terminal residues of t elicits a DnaG-stimulatory effect in vitro that appears to be suppressed in the native t protein.