Discrimination against the Cytosine Analog tC by Escherichia coli DNA Polymerase IV DinB
Journal article, 2011

The cytosine analog 1,3-diaza-2-oxophenothiazine (tC) is a fluorescent nucleotide that forms Watson Crick base pairs with dG. The Klenow fragment of DNA polymerase I (an A-family polymerase) can efficiently bypass tC on the template strand and incorporate deoxyribose-triphosphate-tC into the growing primer terminus. Y-family DNA polymerases are known for their ability to accommodate bulky lesions and modified bases and to replicate beyond such nonstandard DNA structures in a process known as translesion synthesis. We probed the ability of the Escherichia coli Y-family DNA polymerase DinB (Pol IV) to copy DNA containing tC and to incorporate tC into a growing DNA strand. DinB selectively adds dGTP across from tC in template DNA but cannot extend beyond the newly formed G:tC base pair. However, we find that DinB incorporates the tC deoxyribonucleotide triphosphate opposite template G and extends from tC. Therefore, DinB displays asymmetry in terms of its ability to discriminate against the modification of the DNA template compared to the incoming nucleotide. In addition, our finding that DinB (a lesion-bypass DNA polymerase) specifically discriminates against tC in the template strand may suggest that DinB discriminates against template modifications in the major groove of DNA.

major groove

translesion synthesis

tryptophan fluorescence binding assay

primer extension

Author

J. M. Walsh

Northeastern University

I. Bouamaied

University of Southampton

T. Brown

University of Southampton

Marcus Wilhelmsson

Chalmers, Chemical and Biological Engineering, Physical Chemistry

P. J. Beuning

Northeastern University

Journal of Molecular Biology

0022-2836 (ISSN)

Vol. 409 2 89-100

Areas of Advance

Nanoscience and Nanotechnology (2010-2017)

Life Science Engineering (2010-2018)

Subject Categories

Physical Chemistry

Biochemistry and Molecular Biology

Biophysics

DOI

10.1016/j.jmb.2011.03.069

More information

Latest update

2/28/2018