Cloning, characterization and expression analysis of nucleotide metabolism-related genes of mycobacteriophage L5

Bhattacharya, Bidisha; Giri, Nabanita; Mitra, Mahasweta; Gupta, S. K.

doi:10.1111/j.1574-6968.2007.01047.x

Cited by 17 publications

(29 citation statements)

References 35 publications

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“…The rest of the sequence is virtually identical between the two. For phage induction experiments the thermo-inducible lysogenic strain Mycobacterium smegmatis L1c1ts (a temperature-sensitive lysogenic strain of L1 obtained from N. C. Mandal) was used (4,9). The phage L1 is considered identical to L5 except for minor variations.…”

Section: Methodsmentioning

confidence: 99%

The Mycobacteriophage D29 Gene 65 Encodes an Early-Expressed Protein That Functions as a Structure-Specific Nuclease

et al. 2009

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The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to DNA synthesis. One such gene is 65, which encodes a protein belonging to the RecA/DnaB helicase superfamily. In this study a recombinant version of the mycobacteriophage D29 gp65 was functionally characterized. The results indicated that it is not a helicase as predicted but an exonuclease that removes 3 arms from forked structures in an ATP-dependent manner. The gp65 exonuclease acts progressively from the 3 end, until the fork junction is reached. As it goes past, its progress is stalled over a stretch of seven to eight nucleotides immediately downstream of the junction. It efficiently acts on forked structures with single stranded arms. It also acts upon 5 and 3 flaps, though with somewhat relaxed specificity, but not on double-stranded forks. Sequence comparison revealed the presence of a KNRXG motif in the C-terminal half of the protein. This is a conserved element found in the RadA/Sms family of DNA repair proteins. A mutation (R203G) in this motif led to complete loss of nuclease activity. This indicated that KNRXG plays an important role in the nuclease function of not only gp65, but possibly other RadA/Sms family proteins as well. This is the first characterization of a bacteriophagederived RadA/Sms class protein. Given its mode of action, it is very likely that gp65 is involved in processing branched replication intermediates formed during the replication of phage DNA.

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Section: Methodsmentioning

confidence: 99%

The Mycobacteriophage D29 Gene 65 Encodes an Early-Expressed Protein That Functions as a Structure-Specific Nuclease

et al. 2009

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“…Gene 50 of mycobacteriophage D29 and related phages encodes a class II ribonucleotide reductase (RNRII). In a previous study, this protein was biochemically characterized (24). In order to investigate its function, an attempt was made to express this gene in M. smegmatis strain ⌬DRKIN, in which one copy of a 56-kb duplication found in M. smegmatis mc 2 155 is missing (20).…”

Section: Resultsmentioning

confidence: 99%

“…S2 and S3 in the supplemental material). The dinB2 gene of M. smegmatis (MSMEG_2294) (20) and D29 gene 48, which codes for D29Gp48, a thymidylate synthase (TS) (24), were amplified by using specific primers (see Tables S1 and S2 in the supplemental material) and cloned into the multiple-cloning site (MCS) of the pMind vector to generate pSG16 and pSG17, respectively (see Fig. S1 in the supplemental material).…”

Section: Methodsmentioning

confidence: 99%

“…To prepare cell extracts, 20 ml of cell suspension was pelleted at 9,300 ϫ g for 10 min, washed twice with Tris-sorbitol buffer, resuspended in 1 ml of phosphate-buffered saline (pH 7.5), and lysed by sonication. To confirm that the target genes were successfully expressed, Western blot analysis was performed with antisera raised in rabbits, essentially as reported previously (24).…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, dTTP deficiency could specifically lead to what is known as "thymineless death" (TLD), which has been studied extensively in various organisms (40). The genomes of D29 and related phages possess genes encoding not only RNR (Gp50) but also TS (D29Gp48) (24). It was therefore hypothesized that if D29Gp48 is synthesized within the cell along with Gp50, the disparity in the dTTP level relative to the other dNTPs would be corrected, resulting in uninhibited cell growth.…”

Section: Fig 2 Comparison Of Ori/ter Ratios For Chromosomal Dna Isolamentioning

confidence: 99%

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A DinB Ortholog Enables Mycobacterial Growth under dTTP-Limiting Conditions Induced by the Expression of a Mycobacteriophage-Derived Ribonucleotide Reductase Gene

et al. 2016

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Mycobacterium species such as M. smegmatis and M. tuberculosis encode at least two translesion synthesis (TLS) polymerases, DinB1 and DinB2, respectively. Although predicted to be linked to DNA repair, their role in vivo remains enigmatic. M. smegmatis mc 2 155, a strain commonly used to investigate mycobacterial genetics, has two copies of dinB2, the gene that codes for DinB2, by virtue of a 56-kb chromosomal duplication. Expression of a mycobacteriophage D29 gene (gene 50) encoding a class II ribonucleotide reductase in M. smegmatis ⌬DRKIN, a strain derived from mc 2 155 in which one copy of the duplication is lost, resulted in DNA replication defects and growth inhibition. The inhibitory effect could be linked to the deficiency of dTTP that resulted under these circumstances. The selective inhibition observed in the ⌬DRKIN strain was found to be due solely to a reduced dosage of dinB2 in this strain. Mycobacterium bovis, which is closely related to M. tuberculosis, the tuberculosis pathogen, was found to be highly susceptible to gene 50 overexpression. Incidentally, these slow-growing pathogens harbor one copy of dinB2. The results indicate that the induction of a dTTP-limiting state can lead to growth inhibition in mycobacteria, with the effect being maximum in cells deficient in DinB2. IMPORTANCEMycobacterium species, such as M. tuberculosis, the tuberculosis pathogen, are known to encode several Y family DNA polymerases, one of which is DinB2, an ortholog of the DNA repair-related protein DinP of Escherichia coli. Although this protein has been biochemically characterized previously and found to be capable of translesion synthesis in vitro, its in vivo function remains unknown. Using a novel method to induce dTTP deficiency in mycobacteria, we demonstrate that DinB2 can aid mycobacterial survival under such conditions. Apart from unraveling a specific role for the mycobacterial Y family DNA polymerase DinB2 for the first time, this study also paves the way for the development of drugs that can kill mycobacteria by inducing a dTTP-deficient state. Members of the Y family DNA polymerases are capable of translesion synthesis (TLS) that allows them to catalyze the insertion of deoxyribonucleotide triphosphates (dNTPs) opposite potentially lethal replication-blocking lesions (1). The ability of these polymerases to bypass such lesions helps the cell to survive under DNA-damaging conditions. However, survival comes at a cost. Mutations are introduced more frequently than under normal conditions, as these polymerases function in an error-prone manner (2). In Escherichia coli, DinP/DinB (DNA polymerase IV [Pol IV]) and UmuC (DNA Pol V) are the two Y family polymerases that mediate TLS (2). Mutations in the genes that encode UmuC and a related protein, UmuD, result in a UV-nonmutable (umu) phenotype. In contrast, mutation of dinB, the gene that codes for the Pol IV enzyme DinB, does not lead to an apparent phenotype, and therefore, the function of this protein remains enigmatic. The expression of the E. col...

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Crystal structure and enzymatic characterization of thymidylate synthase X from Helicobacter pylori strain SS1

Wang

Chen

et al. 2011

Protein Science

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Thymidylate synthase X (ThyX) catalyzes the methylation of dUMP to form dTMP in bacterial life cycle and is regarded as a promising target for antibiotics discovery. Helicobacter pylori is a human pathogen associated with a number of human diseases. Here, we cloned and purified the ThyX enzyme from H. pylori SS1 strain (HpThyX). The recombinant HpThyX was discovered to exhibit the maximum activity at pH 8.5, and K m values of the two substrates dUMP and CH 2 H 4 folate were determined to be 15.3 6 1.25 lM and 0.35 6 0.18 mM, respectively. The analyzed crystal structure of HpThyX with the cofactor FAD and the substrate dUMP (at 2.31 Å ) revealed that the enzyme was a tetramer bound to four dUMP and four FAD molecules. Different from the catalytic feature of the classical thymidylate synthase (ThyA), N5 atom of the FAD functioned as a nucleophile in the catalytic reaction instead of Ser84 and Ser85 residues. Our current work is expected to help better understand the structural and enzymatic features of HpThyX thus further providing valuable information for anti-H. pylori inhibitor discovery.

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Cloning, characterization and expression analysis of nucleotide metabolism-related genes of mycobacteriophage L5

Cited by 17 publications

References 35 publications

The Mycobacteriophage D29 Gene 65 Encodes an Early-Expressed Protein That Functions as a Structure-Specific Nuclease

The Mycobacteriophage D29 Gene 65 Encodes an Early-Expressed Protein That Functions as a Structure-Specific Nuclease

A DinB Ortholog Enables Mycobacterial Growth under dTTP-Limiting Conditions Induced by the Expression of a Mycobacteriophage-Derived Ribonucleotide Reductase Gene

Crystal structure and enzymatic characterization of thymidylate synthase X from Helicobacter pylori strain SS1

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