Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans SCIE SCOPUS

Cited 49 time in WEB OF SCIENCE Cited 53 time in Scopus
Title
Protein trans-splicing and characterization of a split family B-type DNA polymerase from the hyperthermophilic archaeal parasite Nanoarchaeum equitans
Author(s)
Choi, JJ; Nam, KH; Min, B; Kim, SJ; Soll, D; Kwon, ST
Publication Year
2006-03-10
Abstract
Nanoarchaeum equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two separate genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA polymerase genes were cloned and expressed in Escherichia coli individually, together (for the Neq C), and as a genetically protein splicing-processed form (Neq P). The protein trans-spliced Neq C was obtained using the heating step at 80 degrees C after the co-expression of the two genes. The protein trans-splicing of the N-terminal and C-terminal parts of Neq DNA polymerase was examined in vitro using the purified Neq L and Neq S. The trans-splicing was influenced mainly by temperature, and occurred only at temperatures above 50 degrees C. The trans-splicing reaction was inhibited in the presence of zinc. Neq S has no catalytic activity and Neq L has lower 3'-5' exonuclease activity; whereas Neq C and Neq P have polymerase and 3'-5' exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase that possesses higher proofreading activity. The genetically protein splicing-processed Neq P showed the same properties as the protein trans-spliced Neq C. Our results are the first evidence to show experimentally that natural protein trans-splicing occurs in an archaeal protein, a thermostable protein, and a family B-type DNA polymerase. (c) 2006 Elsevier Ltd. All rights reserved.
ISSN
0022-2836
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/4905
DOI
10.1016/j.jmb.2005.12.036
Bibliographic Citation
JOURNAL OF MOLECULAR BIOLOGY, v.356, no.5, pp.1093 - 1106, 2006
Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
Subject
THERMOAUTOTROPHICUM DELTA-H; IN-VITRO; PHYLUM NANOARCHAEOTA; ZINC ION; INTEIN; GENE; EXPRESSION; SEQUENCE; CLEAVAGE; PCC6803
Keywords
protein trans-splicing; Nanoarchaeum equitans; nanoarchaeon; family B-type DNA polymerase; biochemical characterization
Type
Article
Language
English
Document Type
Article
Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
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