4DTU

RB69 DNA Polymerase Ternary Complex with dGTP Opposite an Abasic Site and ddC/dG as the Penultimate Base-pair

Summary for 4DTU

• Entry DOI: 10.2210/pdb4dtu/pdb
• Related: 4DTJ 4DTM 4DTN 4DTO 4DTP 4DTR 4DTS 4DTX 4DU1 4DU3 4DU4
• Descriptor: DNA polymerase, DNA tempalte, DNA primer, ... (6 entities in total)
• Functional Keywords: abasic site, thf, rb69pol, dgtp, transferase-dna complex, transferase/dna
• Biological source: Enterobacteria phage RB69
• Total number of polymer chains: 3
• Total formula weight: 114598.66

Authors

Xia, S.,Wang, J.,Konigsberg, W.H. (deposition date: 2012-02-21, release date: 2012-06-06, Last modification date: 2024-02-28)

Primary citation

Xia, S.,Vashishtha, A.,Bulkley, D.,Eom, S.H.,Wang, J.,Konigsberg, W.H.
Contribution of Partial Charge Interactions and Base Stacking to the Efficiency of Primer Extension at and beyond Abasic Sites in DNA.
Biochemistry, 51:4922-4931, 2012

PubMed Abstract: During DNA synthesis, base stacking and Watson-Crick (WC) hydrogen bonding increase the stability of nascent base pairs when they are in a ternary complex. To evaluate the contribution of base stacking to the incorporation efficiency of dNTPs when a DNA polymerase encounters an abasic site, we varied the penultimate base pairs (PBs) adjacent to the abasic site using all 16 possible combinations. We then determined pre-steady-state kinetic parameters with an RB69 DNA polymerase variant and solved nine structures of the corresponding ternary complexes. The efficiency of incorporation for incoming dNTPs opposite an abasic site varied between 2- and 210-fold depending on the identity of the PB. We propose that the A rule can be extended to encompass the fact that DNA polymerase can bypass dA/abasic sites more efficiently than other dN/abasic sites. Crystal structures of the ternary complexes show that the surface of the incoming base was stacked against the PB's interface and that the kinetic parameters for dNMP incorporation were consistent with specific features of base stacking, such as surface area and partial charge-charge interactions between the incoming base and the PB. Without a templating nucleotide residue, an incoming dNTP has no base with which it can hydrogen bond and cannot be desolvated, so that these surrounding water molecules become ordered and remain on the PB's surface in the ternary complex. When these water molecules are on top of a hydrophobic patch on the PB, they destabilize the ternary complex, and the incorporation efficiency of incoming dNTPs is reduced.

PubMed: 22630605
DOI: 10.1021/bi300296q

Experimental method

X-RAY DIFFRACTION (1.86 Å)