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

Abstract

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 archaeonThermococcus sibiricuswas expressed inEscherichia coliand 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 groupP212121, with unit-cell parametersa = 58.590,b= 87.540,c= 126.300 Å.

Biotechnological applications of archaeal enzymes from extreme environments

“…The first thermostable ligase was discovered in the bacterium Thermus thermophilus HB8. Most of these enzymes come from thermophilic bacteria, but there are also of them from the hyper/thermophilic archaea Pyrococcus [ 131 – 133 ], Thermococcus [ 133 – 136 ], Hyperthermus butylicus [ 137 ], Methanocaldococcus jannaschii [ 138 ], Methanobacterium thermoautotrophicum [ 139 ], Sulfophobococcus zilligii [ 140 ], Aeropyrum pernix K1 [ 141 ], Archaeoglobus fulgidus [ 142 ], and Sulfolobus [ 143 ] (see Table 9 ). Unlike bacterial DNA ligases, these enzymes require ATP as a cofactor.…”

ATP-dependent DNA ligase fromThermococcussp. 1519 displays a new arrangement of the OB-fold domain

“…This conformation was revealed for PfuLig (Nishida et al, 2006; PDB entry 2cfm; Fig. 1b), AfuLig (Kim et al, 2009; PDB entry 3gde) and TsibLig (Petrova et al, 2012; PDB entry 4eq5).…”

“…Only a few crystal structures of archaeal DNA ligases have been reported: those from the Euryarchaeota P. furiosus (PfuLig; Nishida et al, 2006), Archaeoglobus fulgidus (AfuLig; Kim et al, 2009) and T. sibiricus (TsibLig; Petrova et al, 2012) and the Crenarchaeota Sulfolobus solfataricus (SsoLig; Pascal et al, 2006) and Sulfophobococcus zilligii (Supangat et al, 2010). Previously, we identified and functionally characterized the ATP-dependent DNA ligase from the hyperthermophilic archaeon Thermococcus sp.…”

Thermostable DNA ligases from hyperthermophiles in biotechnology

“…Seventeen DNA ligases have been reported from hyperthermophilic archaea to date, including Methanobacterium thermoautotrophicum ( Sriskanda et al, 2000 ), Thermococcus kodakaraensis ( Nakatani et al, 2000 ), Sulfolobus shibatae ( Lai et al, 2002 ), Aeropyrum pernix ( Jeon and Ishikawa, 2003 ), Thermococcus fumicolans ( Rolland et al, 2004 ), Pyrococcus horikoshii ( Keppetipola and Shuman, 2005 ), P. furiosus ( Nishida et al, 2006 ), Thermococcus sp. NA1 ( Kim et al, 2006 ), Sulfolobus solfataricus ( Pascal et al, 2006 ), Staphylothermus marinus ( Seo et al, 2007 ), Sulfophobococcus zilligii ( Sun et al, 2008 ), Archaeoglobus fulgidus ( Kim et al, 2009 ), Thermococcus sibiricus ( Petrova T. E. et al, 2012 ), Thermococcus sp. 1519 ( Petrova T. et al, 2012 ), Hyperthermus butylicus ( Kim et al, 2013 ), Pyrococcus abyssi ( Oscorbin et al, 2015 ), and Thermococcus barophilus Ch5 ( Shi et al, 2019 ).…”