Expression, purification, crystallization and preliminary crystallographic analysis of a thermostable DNA ligase from the archaeon Thermococcus sibiricus.

DNA ligases join single-strand breaks in double-stranded DNA by catalyzing the formation of a phosphodiester bond between adjacent 5'-phosphate and 3'-hydroxyl termini. Their function is essential to maintain the integrity of the genome in DNA replication, recombination and repair. A recombinant ATP-dependent DNA ligase from the hyperthermophilic anaerobic archaeon Thermococcus sibiricus was expressed in Escherichia coli and purified. Crystals were grown by vapour diffusion using the hanging-drop method with 17%(w/v) PEG 4000 and 8.5%(v/v) 2-propanol as precipitants. A diffraction experiment was performed with a single crystal, which diffracted X-rays to 3.0 Å resolution. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 58.590, b = 87.540, c = 126.300 Å.

[Structural organization and control of expression of the sop-operon of linear plasmid prophage N15]. / Strukturnaia organizatsiia i kontrol' ékspressii sop-operona lineinogo plazmidnogo profaga N15.

Stable inheritance of bacterial chromosomes and low-copy-number plasmids depends on the active partition of replicated molecules between daughter cells. The partition mechanism is well known for circular plasmids F and P1. The mechanism of partition of linear replicons was studied with the example of bacteriophage N15, which persists as a linear plasmid with covalently closed ends on lysogeny, rather than integrating into the Escherichia coli chromosome. Since stable inheritance of N15 is due to the sop operon homologous to sop of the F plasmid, the control of expression of the N15 sop genes was analyzed. The sop promoter (Psop) contains a binding site for bacterial IHF and five CTTTGC copies, which overlap the -35 and -10 elements. The Sop proteins were shown to interact with a Psop-containing DNA fragment in vitro. Transcription of the sop operon is regulated by the Sop proteins: SopA represses Psop, and SopB enhances the repression, having no effect on the promoter activity in the absence of SopA. In N15 lysogenic cells, Psop proved to be repressed. This regulatory mechanism was assumed to ensure production of SopA and SopB in amounts required for the segregation stability of N15 and to neutralize occasional fluctuations of their concentration in the cell.

[Expression regulation of the protelomerase gene of the bacteriophage N15]. / Reguliatsiia ékspresii gena protelomerazy bakteriofaga N15.

The N15 bacteriophage, when in the lysogenic state, does not integrate into the chromosome; in fact, it exists as a linear plasmid with the covalently closed ends. Upon infection, the phage DNA circularizes via its cohesive ends, after which a specific enzyme, the N15 protelomerase, cuts the circular molecule thus generating a linear plasmid with the covalently closed telomeres. Protelomerase generates, as the replication of plasmid prophage proceeds, the hairpin telomeres in replicated molecules. We identified the promoter of the protelomerase gene and demonstrated that it could be repressed presumably due to its binding with 3 tosL sites overlapping the promoter. We also found the transformation efficiency of E. coli cells of linear DNA with hairpin telomeres to be approximately 100-fold lower versus the circular DNA of the same size. At the same time, presence of the N15 prophage or of the protelomerase-expressing vector enhances, in a strain being transformed, the efficiency of its transformation by linear DNA up to a level ensured in transformation by circular plasmids. We believe that protelomerase, while binding with the hairpin telomeres, protects the latter from degradation by cellular nucleases.