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TitleIdentification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme.
Journal, issue, pagesNat Commun, Vol. 8, Page 15075, Year 2017
Publish dateMay 2, 2017
AuthorsJiangyu Yan / Thomas R Beattie / Adriana L Rojas / Kelly Schermerhorn / Tamzin Gristwood / Jonathan C Trinidad / Sonja V Albers / Pietro Roversi / Andrew F Gardner / Nicola G A Abrescia / Stephen D Bell /
PubMed AbstractSince their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family ...Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.
External linksNat Commun / PubMed:28462924 / PubMed Central
MethodsEM (single particle) / X-ray diffraction
Resolution1.351 - 22.8 Å
Structure data

EMDB-3458:
negative-stain volume of Sso DNA PolB1
Method: EM (single particle) / Resolution: 22.8 Å

EMDB-3462:
negative-stain 3D reconstruction of Sso heterotrimeric holo DNA-PolB1
Method: EM (single particle) / Resolution: 20.2 Å

PDB-5n35:
Gadolinium phased PBP2 (SSO6202) at 2.2 Ang
Method: X-RAY DIFFRACTION / Resolution: 2.24 Å

PDB-5n41:
Archaeal DNA polymerase holoenzyme - SSO6202 at 1.35 Ang resolution
Method: X-RAY DIFFRACTION / Resolution: 1.351 Å

Chemicals

ChemComp-GD:
GADOLINIUM ATOM

ChemComp-GOL:
GLYCEROL / Glycerol

ChemComp-NO3:
NITRATE ION / Nitrate

ChemComp-HOH:
WATER / Water

ChemComp-EDO:
1,2-ETHANEDIOL / Ethylene glycol

Source
  • Archaea (unknown)
  • sulfolobus solfataricus (archaea)
KeywordsPOLYMERASE BINDING PROTEIN / PolB1 binding protein 2 / Archaeal DNA Polymerase Holoenzyme / Protein binding / Polymerase-binding protein

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