Format

Send to

Choose Destination
J Mol Biol. 2002 May 31;319(2):359-69.

DNA polymerase theta purified from human cells is a high-fidelity enzyme.

Author information

1
Istituto di Genetica Molecolare IGM-CNR, via Abbiategrasso 207, 27100 Pavia, Italy. maga@igbe.pv.cnr.it

Abstract

With the aim to identify unconventional DNA polymerases from human cells, we have set up a special assay to fractionate HeLa extracts based on the ability (i) to bypass DNA lesions, (ii) to be resistant to aphidicolin and an inhibitory antibody against pol alpha and (iii) to be non-responsive to proliferating cell nuclear antigen. After eight different chromatographic steps, an aphidicolin-resistant DNA polymerase activity was obtained that was able to utilize either undamaged or abasic sites-containing DNA with the same efficiency. Biochemical characterization and immunoblot analysis allowed its identification as the human homologue of DNA polymerase theta (hpol theta), whose cDNA has been cloned by homology with the mus308 gene of Drosophila melanogaster but still awaited detailed biochemical characterization. The purified hpol theta was devoid of detectable helicase activity, possessed a 3'-->5' exonuclease activity and showed biochemical properties clearly distinct from any other eukaryotic DNA polymerase known so far. Misincorporation and fidelity assays showed that: (i) hpol theta was able to catalyze efficiently DNA synthesis past an abasic site; and (ii) hpol theta showed high fidelity. Our findings are discussed in light of the proposed physiological role of hpol theta.

PMID:
12051913
DOI:
10.1016/S0022-2836(02)00325-X
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Elsevier Science
Loading ...
Support Center