Novel DNA ligase with broad nucleotide cofactor specificity from the hyperthermophilic crenarchaeon Sulfophobococcus zilligii: influence of ancestral DNA ligase on cofactor utilization

Abstract

DNA ligases are divided into two groups according to their cofactor requirement to form ligase-adenylate, ATP-dependent DNA ligases and NAD(+)-dependent DNA ligases. The conventional view that archaeal DNA ligases only utilize ATP has recently been disputed with discoveries of dual-specificity DNA ligases (ATP/ADP or ATP/NAD(+)) from the orders Desulfurococcales and Thermococcales. Here, we studied DNA ligase encoded by the hyperthermophilic crenarchaeon Sulfophobococcus zilligii. The ligase exhibited multiple cofactor specificity utilizing ADP and GTP in addition to ATP. The unusual cofactor specificity was confirmed via a DNA ligase nick-closing activity assay using a fluorescein/biotin-labelled oligonucleotide and a radiolabelled oligonucleotide. The exploitation of GTP as a catalytic energy source has not to date been reported in any known DNA ligase. This phenomenon may provide evolutionary evidence of the nucleotide cofactor utilization by DNA ligases. To bolster this hypothesis, we summarize and evaluate previous assertions. We contend that DNA ligase evolution likely started from crenarchaeotal DNA ligases and diverged to eukaryal DNA ligases and euryarchaeotal DNA ligases. Subsequently, the NAD(+)-utilizing property of some euryarchaeotal DNA ligases may have successfully differentiated to bacterial NAD(+)-dependent DNA ligases.

The Hyperthermophilic Euryarchaeon Archaeoglobus fulgidus Repairs Uracil by Single-Nucleotide Replacement

“…Bacterial DNA ligases use NAD and the mammalian enzymes use ATP for adenylation (32). Although there are exceptions (62,71), the archaeal ligases are considered ATP dependent and exhibit sequence homology with eukaryotic enzymes (12), and some of them have also been demonstrated to utilize ADP with similar efficiency (25). This is consistent with our results, which showed that addition of NAD increased the level of repaired DNA to a level somewhat greater than the control level, while addition of ATP and ADP resulted in a Ͼ1-order-of-magnitude increase in the level of the repair product following incubation for 60 min (Fig.…”

Rapid progress of DNA replication studies in Archaea, the third domain of life

“…Further dual co-factor specificity exists in the archaeal DNA ligases, which use both ADP and ATP, as found in the enzymes from A. pernix [166] and Staphylothermus marinus [167]. In the case of Sulfobococcus zilligii, GTP is also the functional cofactor [168]. The DNA ligases from P. horikoshii [169] and P. furiosus [170] have a strict ATP preference.…”

“…Acidithiobacillus ferrooxidans ATP [165] Aeropyrum pernix ATP ADP [166] Ferroplasma acidarmanus ATP [197] Ferroplasma acidophilum ATP NAD + [165] Methanothermobacterium thermoautotrophicum ATP [198] Picrophilus torridus ATP NAD + [165] Pyrococcus horikoshii ATP [169] Pyrococcus furiosus ATP [170] Staphylothermus marinus ATP ADP [167] Sulfophobococcus zilligii ATP ADP GTP [168] Sulfolobus acidocaldarius ATP [165] Sulfolobus shibatae ATP [199] Thermococcus fumicolans ATP NAD + [163] Thermococcus kodakarensis ATP NAD + [162] Thermococcus sp. ATP NAD + [164] Thermococcus sp.…”

Diversity of the DNA Replication System in theArchaeaDomain

“…A thermophilic DNA ligase from M. thermautotrophicus which uses ATP as sole cofactor was characterized in vitro [155]. A characterized DNA ligase from the crenarchaeote Sulfophobococcus zilligii displayed specificity for three cofactors (ATP/NAD + /GTP) [156]. Major structural work in this enzyme has been achieved in P. furiosus and S. solfataricus , and the findings have been reviewed in detail [43].…”