Characterization of the 3' exonuclease subunit DP1 of Methanococcus jannaschii replicative DNA polymerase D.

Abstract:

:The B-subunits associated with the replicative DNA polymerases are conserved from Archaea to humans, whereas the corresponding catalytic subunits are not related. The latter belong to the B and D DNA polymerase families in eukaryotes and archaea, respectively. Sequence analysis places the B-subunits within the calcineurin-like phosphoesterase superfamily. Since residues implicated in metal binding and catalysis are well conserved in archaeal family D DNA polymerases, it has been hypothesized that the B-subunit could be responsible for the 3'-5' proofreading exonuclease activity of these enzymes. To test this hypothesis we expressed Methanococcus jannaschii DP1 (MjaDP1), the B-subunit of DNA polymerase D, in Escherichia coli, and demonstrate that MjaDP1 functions alone as a moderately active, thermostable, Mn2+-dependent 3'-5' exonuclease. The putative polymerase subunit DP2 is not required. The nuclease activity is strongly reduced by single amino acid mutations in the phosphoesterase domain indicating the requirement of this domain for the activity. MjaDP1 acts as a unidirectional, non-processive exonuclease preferring mispaired nucleotides and single-stranded DNA, suggesting that MjaDP1 functions as the proofreading exonuclease of archaeal family D DNA polymerase.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Jokela M,Eskelinen A,Pospiech H,Rouvinen J,Syväoja JE

doi

10.1093/nar/gkh558

keywords:

subject

Has Abstract

pub_date

2004-04-30 00:00:00

pages

2430-40

issue

8

eissn

0305-1048

issn

1362-4962

pii

32/8/2430

journal_volume

32

pub_type

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