4TQS

Ternary complex of Y-family DNA polymerase Dpo4 with (5'S)-8,5'-Cyclo-2'-deoxyguanosine and dCTP

Summary for 4TQS

• Entry DOI: 10.2210/pdb4tqs/pdb
• Descriptor: DNA polymerase IV, DNA (5'-D(*GP*GP*GP*GP*GP*AP*AP*GP*GP*AP*TP*T)-3'), DNA (5'-D(P*TP*CP*AP*CP*(2LF)P*GP*AP*AP*TP*CP*CP*TP*TP*CP*CP*CP*CP*C)-3'), ... (7 entities in total)
• Functional Keywords: y-family dna polymerase, dna damage, cyclopurine, translesion dna synthesis, transferase, transferase-dna complex, transferase/dna
• Biological source: Sulfolobus solfataricus; More
• Total number of polymer chains: 6
• Total formula weight: 102143.35

Authors

Zhao, L. (deposition date: 2014-06-11, release date: 2015-01-14, Last modification date: 2023-12-27)

Primary citation

Xu, W.,Ouellette, A.M.,Wawrzak, Z.,Shriver, S.J.,Anderson, S.M.,Zhao, L.
Kinetic and Structural Mechanisms of (5'S)-8,5'-Cyclo-2'-deoxyguanosine-Induced DNA Replication Stalling.
Biochemistry, 54:639-651, 2015

PubMed 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.

PubMed: 25569151
DOI: 10.1021/bi5014936

Experimental method

X-RAY DIFFRACTION (2.06 Å)