PDBsum entry 4tqs

Transferase/DNA

PDB id: 4tqs

Contents

Protein chains

342 a.a.

DNA/RNA

Ligands

DCP ×2

DOC ×2

Metals

_MG ×4

Waters ×488

PDB id:

4tqs

Name:

Transferase/DNA

Title:

Ternary complex of y-family DNA polymerase dpo4 with (5's)-8,5'-cyclo- 2'-deoxyguanosine and dctp

Structure:

DNA polymerase iv. Chain: a, b. Synonym: pol iv. Engineered: yes. DNA (5'-d( Gp Gp Gp Gp Gp Ap Ap Gp Gp Ap Tp T)-3'). Chain: c, p. Engineered: yes. DNA (5'-d(p Tp Cp Ap Cp (2Lf) p Gp Ap Ap Tp Cp Cp Tp Tp Cp Cp Cp Cp C)-3').

Source:

Sulfolobus solfataricus. Organism_taxid: 273057. Strain: atcc 35092 / dsm 1617 / jcm 11322 / p2. Gene: dbh, dpo4, sso2448. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Synthetic construct. Organism_taxid: 32630.

Resolution:

2.06Å R-factor: 0.231 R-free: 0.290

Authors:

L.Zhao

Key ref:

W.Xu et al. (2015). Kinetic and structural mechanisms of (5'S)-8,5'-cyclo-2'-deoxyguanosine-induced dna replication stalling. Biochemistry, 54, 639-651. PubMed id: 25569151 DOI: 10.1021/bi5014936

Date:

11-Jun-14 Release date: 14-Jan-15

Protein chains

Q97W02  (DPO4_SULSO) -  DNA polymerase IV from Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2)

Seq: 352 a.a.

Struc: 342 a.a.

DNA/RNA chains

G-G-G-G-G-A-A-G-G-A-T-T 12 bases

G-A-A-T-C-C-T-T-C-C-C-C-C 13 bases

G-G-G-G-G-A-A-G-G-A-T-T 12 bases

2LF-G-A-A-T-C-C-T-T-C-C-C-C-C 14 bases

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

DOI no: 10.1021/bi5014936

Biochemistry 54:639-651 (2015)

PubMed id: 25569151

Kinetic and structural mechanisms of (5'S)-8,5'-cyclo-2'-deoxyguanosine-induced dna replication stalling.

W.Xu, A.M.Ouellette, Z.Wawrzak, S.J.Shriver, S.M.Anderson, L.Zhao.

ABSTRACT

The (5'S)-8,5'-cyclo-2'-deoxyguanosine (S-cdG) lesion is produced from reactions of DNA with hydroxyl radicals generated from ionizing radiation or endogenous oxidative metabolisms. An elevated level of S-cdG has been detected in Xeroderma pigmentosum, Cockayne syndrome, breast cancer patients, and aged mice. S-dG blocks DNA replication and transcription in vitro and in human cells and produces mutant replication and transcription products in vitro and in vivo. Major cellular protection against S-dG includes nucleotide excision repair and translesion DNA synthesis. We used kinetic and crystallographic approaches to elucidate the molecular mechanisms of S-cdG-induced DNA replication stalling using model B-family Sulfolobus solfataricus P2 DNA polymerase B1 (Dpo1) and Y-family S. solfataricus P2 DNA polymerase IV (Dpo4). Dpo1 and Dpo4 inefficiently bypassed S-cdG with dCTP preferably incorporated and dTTP (for Dpo4) or dATP (for Dpo1) misincorporated. Pre-steady-state kinetics and crystallographic data mechanistically explained the low-efficiency bypass. For Dpo1, S-cdG attenuated Kd,dNTP,app and kpol. For Dpo4, the S-cdG-adducted duplex caused a 6-fold decrease in Dpo4:DNA binding affinity and significantly reduced the concentration of the productive Dpo4:DNA:dCTP complex. Consistent with the inefficient bypass, crystal structures of Dpo4:DNA(S-cdG):dCTP (error-free) and Dpo4:DNA(S-cdG):dTTP (error-prone) complexes were catalytically incompetent. In the Dpo4:DNA(S-cdG):dTTP structure, S-cdG induced a loop structure and caused an unusual 5'-template base clustering at the active site, providing the first structural evidence of the previously suggested template loop structure that can be induced by a cyclopurine lesion. Together, our results provided mechanistic insights into S-cdG-induced DNA replication stalling.