scholarly journals Biochemical characterization of the Hjc Holliday junction resolvase of Pyrococcus furiosus

2000 ◽  
Vol 28 (22) ◽  
pp. 4544-4551 ◽  
Author(s):  
K. Komori
Keyword(s):  
Biochemical Characterization ◽  
Pyrococcus Furiosus ◽  
Holliday Junction ◽  
Holliday Junction Resolvase

scholarly journals Sequence Specificity and Biochemical Characterization of the RusA Holliday Junction Resolvase ofEscherichia coli

1997 ◽  
Vol 272 (23) ◽  
pp. 14873-14882 ◽  
Author(s):  
Sau N. Chan ◽  
Lynda Harris ◽  
Edward L. Bolt ◽  
Matthew C. Whitby ◽  
Robert G. Lloyd
Keyword(s):  
Biochemical Characterization ◽  
Ofescherichia Coli ◽  
Holliday Junction ◽  
Sequence Specificity ◽  
Holliday Junction Resolvase

scholarly journals Canonical and novel non-canonical activities of the Holliday junction resolvase Yen1

2020 ◽  
Author(s):  
F. Javier Aguado ◽  
Raquel Carreira ◽  
Vanesa Hurtado-Nieves ◽  
Miguel G. Blanco
Keyword(s):  
Budding Yeast ◽  
Holliday Junction ◽  
Conformational Preference ◽  
Alternative Processing ◽  
Holliday Junction Resolvase ◽  
Replication Intermediates

ABSTRACTYen1 and GEN1 are members of the Rad2/XPG family of nucleases that were identified as the first canonical nuclear Holliday junction (HJ) resolvases in budding yeast and humans due to their ability to introduce two symmetric, coordinated incisions on opposite strands of the HJ, yielding nicked DNA products that could be readily ligated. While GEN1 has been extensively characterized in vitro, much less is known about the biochemistry of Yen1. Here, we have performed the first in-depth characterization of purified Yen1. We confirmed that Yen1 resembles GEN1 in many aspects, including range of substrates targeted, position of most incisions they produce or monomeric state in solution. However, we have also observed unexpected alternative processing of substrates, such as nicked HJs and a different conformational preference on intact HJs. Moreover, we demonstrate that Yen1 is endowed with additional nuclease activities, like a nick-specific 5’-3’ exonuclease or HJ arm-chopping that could apparently blur its classification as a canonical HJ resolvase. Despite this, we show that Yen1 fulfills the requirements of a canonical HJ resolvase and hypothesize that its wider array of nuclease activities might contribute to its function in the removal of persistent recombination or replication intermediates.

scholarly journals Biochemical adaptations of two sugar kinases from the hyperthermophilic archaeon Pyrococcus furiosus

2002 ◽  
Vol 366 (1) ◽  
pp. 121-127 ◽  
Author(s):  
Corné H. VERHEES ◽  
Denise G.M. KOOT ◽  
Thijs J.G. ETTEMA ◽  
Cor DIJKEMA ◽  
Willem M. de VOS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
Pyrococcus Furiosus ◽  
Phosphoryl Group ◽  
Hyperthermophilic Archaeon ◽  
High Affinity ◽  
Optimal Activity ◽  
Genes Encoding ◽  
Modified Embden Meyerhof Pathway

The hyperthermophilic archaeon Pyrococcus furiosus possesses a modified Embden—Meyerhof pathway, including an unusual ADP-dependent glucokinase (ADP-GLK) and an ADP-dependent phosphofructokinase. In the present study, we report the characterization of a P. furiosus galactokinase (GALK) and its comparison with the P. furiosus ADP-GLK. The pyrococcal genes encoding the ADP-GLK and GALK were functionally expressed in Escherichia coli, and the proteins were subsequently purified to homogeneity. Both enzymes are specific kinases with an optimal activity at approx. 90°C. Biochemical characterization of these enzymes confirmed that the ADP-GLK is unable to use ATP as the phosphoryl group donor, but revealed that GALK is ATP-dependent and has an extremely high affinity for ATP. There is a discussion about whether the unusual features of these two classes of kinases might reflect adaptations to a relatively low intracellular ATP concentration in the hyperthermophilic archaeon P. furiosus.

scholarly journals Molecular and Biochemical Characterization of a Distinct Type of Fructose-1,6-Bisphosphatase from Pyrococcus furiosus

2002 ◽  
Vol 184 (12) ◽  
pp. 3401-3405 ◽  
Author(s):  
Corné H. Verhees ◽  
Jasper Akerboom ◽  
Emile Schiltz ◽  
Willem M. de Vos ◽  
John van der Oost
Keyword(s):  
Biochemical Characterization ◽  
Pyrococcus Furiosus ◽  
Distinct Type ◽  
Sequence Motifs ◽  
Conserved Sequence ◽  
New Family ◽  
Fructose 1,6 Bisphosphatase ◽  
Conserved Sequence Motifs ◽  
Fructose 1,6 Bisphosphate

ABSTRACT The Pyrococcus furiosus fbpA gene was cloned and expressed in Escherichia coli, and the fructose-1,6-bisphosphatase produced was subsequently purified and characterized. The dimeric enzyme showed a preference for fructose-1,6-bisphosphate, with a Km of 0.32 mM and a V max of 12.2 U/mg. The P. furiosus fructose-1,6-bisphosphatase was strongly inhibited by Li+ (50% inhibitory concentration, 1 mM). Based on the presence of conserved sequence motifs and the substrate specificity of the P. furiosus fructose-1,6-bisphosphatase, we propose that this enzyme belongs to a new family, class IV fructose-1,6-bisphosphatase.

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