Isolation and characterization of a split B-type DNA polymerase from the archaeon Methanobacterium thermoautotrophicum deltaH.

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TitleIsolation and characterization of a split B-type DNA polymerase from the archaeon Methanobacterium thermoautotrophicum deltaH.
Publication TypeJournal Article
Year of Publication1999
AuthorsKelman, Z, Pietrokovski, S, Hurwitz, J
JournalJ Biol Chem
Volume274
Issue40
Pagination28751-61
Date Published1999 Oct 1
ISSN0021-9258
KeywordsAmino Acid Sequence, Base Sequence, Chromatography, Affinity, Cloning, Molecular, DNA Polymerase beta, DNA Replication, DNA-Binding Proteins, Electrophoresis, Polyacrylamide Gel, Methanobacterium, Molecular Sequence Data, Oligonucleotides, Recombinant Proteins
Abstract

We describe here the isolation and characterization of a B-type DNA polymerase (PolB) from the archaeon Methanobacterium thermoautotrophicum DeltaH. Uniquely, the catalytic domains of M. thermoautotrophicum PolB are encoded from two different genes, a feature that has not been observed as yet in other polymerases. The two genes were cloned, and the proteins were overexpressed in Escherichia coli and purified individually and as a complex. We demonstrate that both polypeptides are needed to form the active polymerase. Similar to other polymerases constituting the B-type family, PolB possesses both polymerase and 3'-5' exonuclease activities. We found that a homolog of replication protein A from M. thermoautotrophicum inhibits the PolB activity. The inhibition of DNA synthesis by replication protein A from M. thermoautotrophicum can be relieved by the addition of M. thermoautotrophicum homologs of replication factor C and proliferating cell nuclear antigen. The possible roles of PolB in M. thermoautotrophicum replication are discussed.

Alternate JournalJ. Biol. Chem.
PubMed ID10497247
Grant ListGM 38559 / GM / NIGMS NIH HHS / United States