Amino acid substitutions at conserved tyrosine 52 alter fidelity and bypass efficiency of human DNA polymerase eta.

Abstract:

DNA polymerase eta (Pol eta) is a member of a new class of DNA polymerases that is able to copy DNA containing damaged nucleotides. These polymerases are highly error-prone during copying of unaltered DNA templates. We analyzed the relationship between bypass efficiency and fidelity of DNA synthesis by introducing substitutions for Tyr-52, a highly conserved amino acid, within the human DNA polymerase eta (hPol eta) finger domain. Most substitutions for Tyr-52 caused reduction in bypass of UV-associated damage, measured by the ability to rescue the viability of UV-sensitive yeast cells at a high UV dose. For most mutants, the reduction in bypass ability paralleled the reduction in polymerization activity. Interestingly, the hPol eta Y52E mutant exhibited a greater reduction in bypass efficiency than polymerization activity. The reduction in bypass efficiency was accompanied by an up to 11-fold increase in the incorporation of complementary nucleotides relative to non-complementary nucleotides. The fidelity of DNA synthesis, measured by copying a gapped M13 DNA template in vitro, was also enhanced as much as 15-fold; the enhancement resulted from a decrease in transitions, which were relatively frequent, and a large decrease in transversions. Our demonstration that an amino acid substitution within the active site enhances the fidelity of DNA synthesis by hPol eta, one of the most inaccurate of DNA polymerases, supports the hypothesis that even error-prone DNA polymerases function in base selection.

Polymerases:

Topics:

Mutational Analysis, Health/Disease, Kinetic Parameters, Nucleotide Analogs / Template Lesions, Exonuclease Activity, Source / Purification

Note:

Mutants of hpol eta had less bypass activity than the wild type. Mutants Y52D and Y52E had only 10% of the bypass activity as the wild type whereas Y52H had about 55% of the bypass activity as the wild type.

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Human Pol eta Glick E2003 Template lesions Bypasses Reaction: Nucleotide incorporation; Substrate: dNTPs; DNA lesion: TT Cyclobutane Pyrimidine Dimer
Human Pol eta Glick E2003 KM 1.7n/a Reaction: Nucleotide incorporation; Substrate: dATP
Human Pol eta Glick E2003 KM 55n/a Reaction: Nucleotide incorporation; Substrate: dCTP
Human Pol eta Glick E2003 KM 3.2n/a Reaction: Nucleotide incorporation; Substrate: dGTP
Human Pol eta Glick E2003 KM 38n/a Reaction: Nucleotide incorporation; Substrate: dTTP
Human Pol eta Glick E2003 Vmax 4 /minute Reaction: Nucleotide incorporation; Substrate: dATP
Human Pol eta Glick E2003 Vmax 2.2 /minute Reaction: Nucleotide incorporation; Substrate: dCTP
Human Pol eta Glick E2003 Vmax 3.6 /minute Reaction: Nucleotide incorporation; Substrate: dGTP
Human Pol eta Glick E2003 Vmax 2.1 /minute Reaction: Nucleotide incorporation; Substrate: dTTP
Human Pol eta Glick E2003 Associated condition UV sensitivity
Pol eta Y52E Glick E2003 3-5' Exonuclease (proofreading) No
Pol eta Y52E Glick E2003 Cloned or native Cloned in yeast
Pol eta Y52E Glick E2003 5-3' Exonuclease No
Pol eta Y52E Glick E2003 Template lesions Bypasses Reaction: Nucleotide incorporation; Substrate: dNTPs; DNA lesion: TT Cyclobutane Pyrimidine Dimer
Pol eta Y52E Glick E2003 KM 3129n/a Reaction: Nucleotide incorporation; Substrate: dTTP
Pol eta Y52E Glick E2003 KM 32n/a Reaction: Nucleotide incorporation; Substrate: dGTP
Pol eta Y52E Glick E2003 KM 448n/a Reaction: Nucleotide incorporation; Substrate: dCTP
Pol eta Y52E Glick E2003 KM 3.93n/a Reaction: Nucleotide incorporation; Substrate: dATP
Pol eta Y52E Glick E2003 Vmax 1.3 /minute Reaction: Nucleotide incorporation; Substrate: dATP
Pol eta Y52E Glick E2003 Vmax 0.5 /minute Reaction: Nucleotide incorporation; Substrate: dCTP
Pol eta Y52E Glick E2003 Vmax 3.5 /minute Reaction: Nucleotide incorporation; Substrate: dGTP
Pol eta Y52E Glick E2003 Vmax 2.1 /minute Reaction: Nucleotide incorporation; Substrate: dTTP
Pol eta Y52E Glick E2003 Associated condition UV sensitivity
Pol eta Y52D Glick E2003 Cloned or native Cloned in yeast
Pol eta Y52D Glick E2003 Template lesions Bypasses Reaction: Nucleotide incorporation; Substrate: dNTPs; DNA lesion: TT Cyclobutane Pyrimidine Dimer
Pol eta Y52D Glick E2003 Associated condition UV sensitivity
Pol eta Y52H Glick E2003 Cloned or native Cloned in yeast
Pol eta Y52H Glick E2003 Template lesions Bypasses Reaction: Nucleotide incorporation; Substrate: dNTPs; DNA lesion: TT Cyclobutane Pyrimidine Dimer
Pol eta Y52H Glick E2003 Associated condition UV sensitivity
Pol eta Y52F Glick E2003 3-5' Exonuclease (proofreading) No
Pol eta Y52F Glick E2003 Cloned or native Cloned in yeast
Pol eta Y52F Glick E2003 5-3' Exonuclease No
Pol eta Y52F Glick E2003 Template lesions Bypasses Reaction: Nucleotide incorporation; Substrate: dNTPs; DNA lesion: TT Cyclobutane Pyrimidine Dimer
Pol eta Y52F Glick E2003 Associated condition UV sensitivity
Pol eta Y52A Glick E2003 3-5' Exonuclease (proofreading) No
Pol eta Y52A Glick E2003 Cloned or native Cloned in yeast
Pol eta Y52A Glick E2003 5-3' Exonuclease No
Pol eta Y52A Glick E2003 Template lesions Bypasses Reaction: Nucleotide incorporation; Substrate: dNTPs; DNA lesion: TT Cyclobutane Pyrimidine Dimer
Pol eta Y52A Glick E2003 Associated condition UV sensitivity

Entry validated by:

Log in to edit reference All References

Using Polbase tables:

Sorting:

Tables may be sorted by clicking on any of the column titles. A second click reverses the sort order. <Ctrl> + click on the column titles to sort by more than one column (e.g. family then name).

Filtering:

It is also possible to filter the table by typing into the search box above the table. This will instantly hide lines from the table that do not contain your search text.