DNA double-strand breaks (DSBs) can be repaired either via homologous recombination (HR) or nonhomologous end-joining (NHEJ). Both pathways are operative in eukaryotes, but bacteria had been thought to rely on HR alone. Here we provide direct evidence that mycobacteria have a robust NHEJ pathway that requires Ku and a specialized polyfunctional ATP-dependent DNA ligase (LigD). NHEJ of blunt-end and complementary 5'-overhang DSBs is highly mutagenic ( approximately 50% error rate). Analysis of the recombination junctions ensuing from individual NHEJ events highlighted the participation of several DNA end-remodeling activities, including template-dependent fill-in of 5' overhangs, nontemplated addition of single nucleotides at blunt ends, and nucleolytic resection. LigD itself has the template-dependent and template-independent polymerase functions in vitro that compose the molecular signatures of NHEJ in vivo. Another ATP-dependent DNA ligase (LigC) provides a backup mechanism for LigD-independent error-prone repair of blunt-end DSBs. We speculate that NHEJ allows mycobacteria to evade genotoxic host defense.
Mycobacterium tuberculosis encodes an NAD ؉ -dependent DNA ligase (LigA) plus three distinct ATP-dependent ligase homologs (LigB, LigC, and LigD). Here we purify and characterize the multiple DNA ligase enzymes of mycobacteria and probe genetically whether the ATP-dependent ligases are required for growth of M. tuberculosis. We find significant differences in the reactivity of mycobacterial ligases with a nicked DNA substrate, whereby LigA and LigB display vigorous nick sealing activity in the presence of NAD ؉ and ATP, respectively, whereas LigC and LigD, which have ATPspecific adenylyltransferase activity, display weak nick joining activity and generate high levels of the DNA- DNA ligases are grouped into two families, ATP-dependent ligases and NAD ϩ -dependent ligases, according to their nucleotide substrate requirement (1, 2). The ligase reaction entails three nucleotidyl transfer steps (1). In the first step, attack by ligase on the ␣ phosphorus of ATP or NAD ϩ results in release of PP i or NMN and formation of a covalent ligase-adenylate intermediate. In the second step, the AMP is transferred to the 5Ј end of the 5Ј phosphate-terminated DNA strand to form DNA-adenylate (AppDNA). In the third step, ligase catalyzes attack by a DNA 3Ј-OH on DNA-adenylate to join the two polynucleotides and liberate AMP. ATP-dependent DNA ligases are found in all three domains of life (Bacteria, Archaea, and Eukarya), whereas the NAD ϩ -dependent enzymes are present only in bacteria and entomopoxviruses (1-7).All known bacteria encode a highly conserved NAD ϩ -dependent DNA ligase (LigA). A conditional mutant of Escherichia coli LigA results in growth arrest at the restrictive temperature; thus LigA is essential in E. coli (8,9). LigA is also essential in Salmonella typhimurium, Bacillus subtilis, and Staphylococcus aureus (10 -12). Some bacteria, including E. coli, S. typhimurium, Shigella flexneri, Yersinia pestis, and Pseudomonas putida, have a second NAD ϩ -dependent ligase (13), the function of which is not known.The presumption that bacteria encode only NAD ϩ -dependent DNA ligases was overturned by the demonstration in 1997 of an ATP-dependent ligase in the respiratory pathogen Haemophilus influenzae (5). The 268-amino acid (aa) 1 H. influenzae ligase consists of a minimized catalytic domain. ATP-dependent ligase homologues coexist with NAD ϩ -dependent enzymes in several other bacterial species, including major human pathogens such as Neisseria meningitidis, Y. pestis, Vibrio cholerae, Pseudomonas aeruginosa, and Mycobacterium tuberculosis (3,14,15).Remarkably, M. tuberculosis encodes three distinct ATP-dependent ligase homologues (LigB, LigC, and LigD) plus an NAD ϩ -dependent ligase (LigA) (Fig. 1) (16). To begin to understand the rationale for this plethora of ligases in a single bacterium, we have produced and characterized recombinant versions of the mycobacterial DNA ligase enzymes and probed genetically the essentiality or dispensability of the ATP-dependent ligases for growth of M. tuberculosis. EXPERIMENTAL ...
RNA triphosphatase catalyzes the first step in mRNA capping. The RNA triphosphatases of fungi and protozoa are structurally and mechanistically unrelated to the analogous mammalian enzyme, a situation that recommends RNA triphosphatase as an anti-infective target. Fungal and protozoan RNA triphosphatases belong to a family of metal-dependent phosphohydrolases exemplified by yeast Cet1. The Cet1 active site is unusually complex and located within a topologically closed hydrophilic b-barrel (the triphosphate tunnel). Here we probe the active site of Plasmodium falciparum RNA triphosphatase by targeted mutagenesis and thereby identify eight residues essential for catalysis. The functional data engender an improved structural alignment in which the Plasmodium counterparts of the Cet1 tunnel strands and active-site functional groups are located with confidence. We gain insight into the evolution of the Cet1-like triphosphatase family by noting that the heretofore unique tertiary structure and active site of Cet1 are recapitulated in recently deposited structures of proteins from Pyrococcus (PBD 1YEM) and Vibrio (PDB 2ACA). The latter proteins exemplify a CYTH domain found in CyaB-like adenylate cyclases and mammalian thiamine triphosphatase. We conclude that the tunnel fold first described for Cet1 is the prototype of a larger enzyme superfamily that includes the CYTH branch. This superfamily, which we name ''triphosphate tunnel metalloenzyme,'' is distributed widely among bacterial, archaeal, and eukaryal taxa. It is now clear that Cet1-like RNA triphosphatases did not arise de novo in unicellular eukarya in tandem with the emergence of caps as the defining feature of eukaryotic mRNA. They likely evolved by incremental changes in an ancestral tunnel enzyme that conferred specificity for RNA 59-end processing.
Paramecium bursaria chlorella virus 1 (PBCV-1) elicits a lytic infection of its unicellular green alga host. The 330-kbp viral genome has been sequenced, yet little is known about how viral mRNAs are synthesized and processed. PBCV-1 encodes its own mRNA guanylyltransferase, which catalyzes the addition of GMP to the 5 diphosphate end of RNA to form a GpppN cap structure. Here we report that PBCV-1 encodes a separate RNA triphosphatase (RTP) that catalyzes the initial step in cap synthesis: hydrolysis of the ␥-phosphate of triphosphate-terminated RNA to generate an RNA diphosphate end. We exploit a yeast-based genetic system to show that Chlorella virus RTP can function as a cap-forming enzyme in vivo. The 193-amino-acid Chlorella virus RTP is the smallest member of a family of metal-dependent phosphohydrolases that includes the RNA triphosphatases of fungi and other large eukaryotic DNA viruses (poxviruses, African swine fever virus, and baculoviruses). Chlorella virus RTP is more similar in structure to the yeast RNA triphosphatases than to the enzymes of metazoan DNA viruses. Indeed, PBCV-1 is unique among DNA viruses in that the triphosphatase and guanylyltransferase steps of cap formation are catalyzed by separate viral enzymes instead of a single viral polypeptide with multiple catalytic domains.The m7GpppN cap structure of eukaryotic mRNA is formed cotranscriptionally by three enzymatic reactions: (i) the 5Ј triphosphate end of the nascent RNA is hydrolyzed to a diphosphate by RNA triphosphatase (RTP), (ii) the diphosphate end is capped with GMP by GTP:RNA guanylyltransferase, and (iii) the GpppN cap is methylated by S-adenosylmethionine: RNA (guanine-N7) methyltransferase (27). DNA viruses have evolved diverse capping strategies. The mRNAs of papovaviruses, parvoviruses, adenoviruses, and herpesviruses are transcribed in the nucleus by RNA polymerase II (Pol II), and their 5Ј ends are modified by the host cell's capping and methylating enzymes. However, vaccinia virus and other poxviruses, which replicate in the cytoplasm, encode and encapsidate with the virus particle a multisubunit RNA polymerase and a complete mRNA capping apparatus (26). African swine fever virus (ASFV), which has a cytoplasmic replication phase, also encodes and encapsidates an RNA polymerase and mRNA capping enzymes (24). Baculoviruses, which replicate in the nucleus of insect cells, use Pol II to transcribe early genes, then switch at later times to a virus-encoded transcription system that includes an RNA polymerase and two cap-forming activities-RTP and RNA guanylyltransferase (4,5,14). Paramecium bursaria chlorella virus 1 (PBCV-1) encodes an RNA guanylyltransferase (7), but it is not clear whether it encodes an RNA polymerase and additional mRNA-processing activities.The triphosphatase, guanylyltransferase, and methyltransferase components of the capping apparatus are organized differently in these DNA virus systems. The triphosphatase, guanylyltransferase, and methyltransferase active sites of the vaccinia virus capping enzym...
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