4ECX: Human Dna Polymerase Eta - Dna Ternary Complex: Reaction In The At Crystal At Ph 7.0 For 300 Sec

Citation:
Abstract
DNA synthesis has been extensively studied, but the chemical reaction itself has not been visualized. Here we follow the course of phosphodiester bond formation using time-resolved X-ray crystallography. Native human DNA polymerase eta, DNA and dATP were co-crystallized at pH 6.0 without Mg(2+). The polymerization reaction was initiated by exposing crystals to 1 mM Mg(2+) at pH 7.0, and stopped by freezing at desired time points for structural analysis. The substrates and two Mg(2+) ions are aligned within 40 s, but the bond formation is not evident until 80 s. From 80 to 300 s structures show a mixture of decreasing substrate and increasing product of the nucleotidyl-transfer reaction. Transient electron densities indicate that deprotonation and an accompanying C2'-endo to C3'-endo conversion of the nucleophile 3'-OH are rate limiting. A third Mg(2+) ion, which arrives with the new bond and stabilizes the intermediate state, may be an unappreciated feature of the two-metal-ion mechanism.
PDB ID: 4ECXDownload
MMDB ID: 101226
PDB Deposition Date: 2012/3/26
Updated in MMDB: 2012/08
Experimental Method:
x-ray diffraction
Resolution: 1.74  Å
Source Organism:
synthetic construct
Similar Structures:
Biological Unit for 4ECX: trimeric; determined by author and by software (PISA)
Molecular Components in 4ECX
Label Count Molecule
Protein (1 molecule)
1
DNA Polymerase ETA(Gene symbol: POLH)
Molecule annotation
Nucleotides(2 molecules)
1
DNA (5'-d(*cp*ap*tp*tp*ap*tp*gp*ap*cp*gp*cp*t)-3')
Molecule annotation
1
DNA (5'-d(*ap*gp*cp*gp*tp*cp*ap*tp*a)-3')
Molecule annotation
Chemicals (8 molecules)
1
3
2
1
3
1
4
1
5
2
* Click molecule labels to explore molecular sequence information.

Citing MMDB
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