Cell Cycle-Dependent Localization and Properties of a Second Mitochondrial DNA Ligase in Crithidia fasciculata

The mitochondrial DNA in kinetoplastid protozoa is contained in a single highly condensed structure consisting of thousands of minicircles and approximately 25 maxicircles. The disk-shaped structure is termed kinetoplast DNA (kDNA) and is located in the mitochondrial matrix near the basal body. We have previously identified a mitochondrial DNA ligase (LIG kβ) in the trypanosomatid Crithidia fasciculata that localizes to antipodal sites flanking the kDNA disk where several other replication proteins are localized. We describe here a second mitochondrial DNA ligase (LIG kα). LIG kα localizes to the kinetoplast primarily in cells that have completed mitosis and contain either a dividing kinetoplast or two newly divided kinetoplasts. Essentially all dividing or newly divided kinetoplasts show localization of LIG kα. The ligase is present on both faces of the kDNA disk and at a high level in the kinetoflagellar zone of the mitochondrial matrix. Cells containing a single nucleus show localization of the LIG kα to the kDNA but at a much lower frequency. The mRNA level of LIG kα varies during the cell cycle out of phase with that of LIG kβ. LIG kα transcript levels are maximal during the phase when cells contain two nuclei, whereas LIG kβ transcript levels are maximal during S phase. The LIG kα protein decays with a half-life of 100 min in the absence of protein synthesis. The periodic expression of the LIG kα transcript and the instability of the LIG kα protein suggest a possible role of the ligase in regulating minicircle replication

Two-step synthesis and hydrolysis of cyclic di-AMP in Mycobacterium tuberculosis.

Cyclic di-AMP is a recently discovered signaling molecule which regulates various aspects of bacterial physiology and virulence. Here we report the characterization of c-di-AMP synthesizing and hydrolyzing proteins from Mycobacterium tuberculosis. Recombinant Rv3586 (MtbDisA) can synthesize c-di-AMP from ATP through the diadenylate cyclase activity. Detailed biochemical characterization of the protein revealed that the diadenylate cyclase (DAC) activity is allosterically regulated by ATP. We have identified the intermediates of the DAC reaction and propose a two-step synthesis of c-di-AMP from ATP/ADP. MtbDisA also possesses ATPase activity which is suppressed in the presence of the DAC activity. Investigations by liquid chromatography -electrospray ionization mass spectrometry have detected multimeric forms of c-di-AMP which have implications for the regulation of c-di-AMP cellular concentration and various pathways regulated by the dinucleotide. We have identified Rv2837c (MtbPDE) to have c-di-AMP specific phosphodiesterase activity. It hydrolyzes c-di-AMP to 5'-AMP in two steps. First, it linearizes c-di-AMP into pApA which is further hydrolyzed to 5'-AMP. MtbPDE is novel compared to c-di-AMP specific phosphodiesterase, YybT (or GdpP) in being a soluble protein and hydrolyzing c-di-AMP to 5'-AMP. Our results suggest that the cellular concentration of c-di-AMP can be regulated by ATP concentration as well as the hydrolysis by MtbPDE

The second messenger cyclic di-AMP negatively regulates the expression of Mycobacterium smegmatis recA and attenuates DNA strand exchange through binding to the C-terminal motif of mycobacterial RecA proteins

Cyclic di-GMP and cyclic di-AMP are second messengers produced by a wide variety of bacteria. They influence bacterial cell survival, biofilm formation, virulence and bacteria-host interactions. However, many of their cellular targets and biological effects are yet to be determined. A chemical proteomics approach revealed that Mycobacterium smegmatis RecA (MsRecA) possesses a high-affinity cyclic di-AMP binding activity. We further demonstrate that both cyclic di-AMP and cyclic di-GMP bind specifically to the C-terminal motif of MsRecA and Mycobacterium tuberculosis RecA (MtRecA). Escherichia coli RecA (EcRecA) was devoid of cyclic di-AMP binding but have cyclic di-GMP binding activity. Notably, cyclic di-AMP attenuates the DNA strand exchange promoted by MsRecA as well as MtRecA through the disassembly of RecA nucleoprotein filaments. However, the structure and DNA strand exchange activity of EcRecA nucleoprotein filaments remain largely unaffected. Furthermore, M. smegmatis disA cells were found to have undetectable RecA levels due to the translational repression of recA mRNA. Consequently, the Delta disA mutant exhibited enhanced sensitivity to DNA-damaging agents. Altogether, this study points out the importance of sequence diversity among recA genes, the role(s) of cyclic di-AMP and reveals a new mode of negative regulation of recA gene expression, DNA repair and homologous recombination in mycobacteria

Hydrolysis of c-di-AMP by MtbPDE.

<p>The reaction mixtures of PDE assay were separated by reverse phase LC and the products were detected by measuring absorbance at 260–4 min have been shown here. The peaks have been labeled with the name of the eluted species. Number on the peak is the retention time of the species. (A) shows the hydrolysis product of c-di-AMP by MtbPDE. Inset shows the control reaction without the enzyme. (B) pApA was hydrolyzed by MtbPDE to AMP. Inset shows the control reaction without MtbPDE. (C) MtbPDE hydrolyzes ApA to AMP and adenosine (D) Hydrolysis of c-di-GMP by MtbPDE to 5′-GMP. Inset shows the control reaction without MtbPDE. (E) Mutant protein D130AH131A does not hydrolyze c-di-AMP.</p

Reaction intermediate ‘I’ was determined to be pppApA.

<p>(A) LC-ESI-MS/MS spectrum of [M+H]⁺ precursor ion m/z 837.05 (reaction intermediate ‘I’). (B) Figure depicting the interpretation of the fragmentation as observed in (A).</p

MtbDisA synthesizes c-di-AMP through two intermediates.

<p>DAC assay was done in 10 µl reaction mixtures containing 25 mM Tris-HCl (pH 8.5), 0.6 mM MnCl₂, 300 µM ATP and 2 µM of MtbDisA and incubated at 37°C. The reaction mixture was subjected to reverse phase LC and the products were detected with UV (λ 260 nm). The absorption chromatograms with peaks labeled with the corresponding species have been shown. (A) Peak for only ATP is detected in the control reaction (without enzyme). ATP is converted into c-di-AMP in the presence of MtbDisA as can be seen in (B). Two more peaks are detected in ‘B’ which are the intermediates of the reaction and are labeled ‘I’ and ‘II’. In (B) the DAC reaction was terminated after 5 min whereas in (C) and (D) the reaction was continued for 10 and 60 min respectively. Peak for ADP has been labeled.</p

Detection of the intermediate ‘I’ of the DAC assay.

<p>(A) LC - ESI -MS spectrum showing the peaks corresponding to proton and manganese ion adducts of the intermediate ‘I’ of molecular mass 836 Da. (B) Expansion of the region, m/z 442–452 of the spectrum shown in (A): m/z values and intensity distribution of isotope peaks indicating doubly charged manganese ion adduct of ‘I’.</p