PDBsum entry 3f1v

Transferase

PDB id: 3f1v

Contents

Protein chains

366 a.a. *

Metals

_CA ×2

_CL

Waters ×378

* Residue conservation analysis

PDB id:

3f1v

Name:

Transferase

Title:

E. Coli beta sliding clamp, 148-153 ala mutant

Structure:

DNA polymerase iii subunit beta. Chain: a, b. Engineered: yes. Mutation: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: dnan, b3701, jw3678. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.77Å R-factor: 0.219 R-free: 0.282

Authors:

V.Cody

Key ref:

J.M.Heltzel et al. (2009). Sliding clamp-DNA interactions are required for viability and contribute to DNA polymerase management in Escherichia coli. J Mol Biol, 387, 74-91. PubMed id: 19361435

Date:

28-Oct-08 Release date: 01-Sep-09

Protein chains

P0A988  (DPO3B_ECOLI) -  Beta sliding clamp from Escherichia coli (strain K12)

Seq: 366 a.a.

Struc: 366 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.7.7.7 - DNA-directed Dna polymerase.
• Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

J Mol Biol 387:74-91 (2009)

PubMed id: 19361435

Sliding clamp-DNA interactions are required for viability and contribute to DNA polymerase management in Escherichia coli.

J.M.Heltzel, S.K.Scouten Ponticelli, L.H.Sanders, J.M.Duzen, V.Cody, J.Pace, E.H.Snell, M.D.Sutton.

ABSTRACT

Sliding clamp proteins topologically encircle DNA and play vital roles in coordinating the actions of various DNA replication, repair, and damage tolerance proteins. At least three distinct surfaces of the Escherichia coli beta clamp interact physically with the DNA that it topologically encircles. We utilized mutant beta clamp proteins bearing G66E and G174A substitutions (beta159), affecting the single-stranded DNA-binding region, or poly-Ala substitutions in place of residues 148-HQDVR-152 (beta(148-152)), affecting the double-stranded DNA binding region, to determine the biological relevance of clamp-DNA interactions. As part of this work, we solved the X-ray crystal structure of beta(148-152), which verified that the poly-Ala substitutions failed to significantly alter the tertiary structure of the clamp. Based on functional assays, both beta159 and beta(148-152) were impaired for loading and retention on a linear primed DNA in vitro. In the case of beta(148-152), this defect was not due to altered interactions with the DnaX clamp loader, but rather was the result of impaired beta(148-152)-DNA interactions. Once loaded, beta(148-152) was proficient for DNA polymerase III (Pol III) replication in vitro. In contrast, beta(148-152) was severely impaired for Pol II and Pol IV replication and was similarly impaired for direct physical interactions with these Pols. Despite its ability to support Pol III replication in vitro, beta(148-152) was unable to support viability of E. coli. Nevertheless, physiological levels of beta(148-152) expressed from a plasmid efficiently complemented the temperature-sensitive growth phenotype of a strain expressing beta159 (dnaN159), provided that Pol II and Pol IV were inactivated. Although this strain was impaired for Pol V-dependent mutagenesis, inactivation of Pol II and Pol IV restored the Pol V mutator phenotype. Taken together, these results support a model in which a sophisticated combination of competitive clamp-DNA, clamp-partner, and partner-DNA interactions serve to manage the actions of the different E. coli Pols in vivo.