This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Google Scholar
Right arrow Articles by Lee, S.-J.
Right arrow Articles by Richardson, C. C.
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, S.-J.
Right arrow Articles by Richardson, C. C.

 Previous Article  |  Next Article 

Journal of Virology, September 2009, p. 8418-8427, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00855-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Rescue of Bacteriophage T7 DNA Polymerase of Low Processivity by Suppressor Mutations Affecting Gene 3 Endonuclease {triangledown}

Seung-Joo Lee, Kajal Chowdhury,{dagger} Stanley Tabor, and Charles C. Richardson*

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115

Received 27 April 2009/ Accepted 11 June 2009

The DNA polymerase encoded by gene 5 (gp5) of bacteriophage T7 has low processivity, dissociating after the incorporation of a few nucleotides. Upon binding to its processivity factor, Escherichia coli thioredoxin (Trx), the processivity is increased to approximately 800 nucleotides per binding event. Several interactions between gp5/Trx and DNA are required for processive DNA synthesis. A basic region in T7 DNA polymerase (residues K587, K589, R590, and R591) is located in proximity to the 5' overhang of the template strand. Replacement of these residues with asparagines results in a threefold reduction of the polymerization activity on primed M13 single-stranded DNA. The altered gp5/Trx exhibits a 10-fold reduction in its ability to support growth of T7 phage lacking gene 5. However, T7 phages that grow at a similar rate provided with either wild-type or altered polymerase emerge. Most of the suppressor phages contain genetic changes in or around the coding region for gene 3, an endonuclease. Altered gene 3 proteins derived from suppressor strains show reduced catalytic activity and are inefficient in complementing growth of T7 phage lacking gene 3. Results from this study reveal that defects in processivity of DNA polymerase can be suppressed by reducing endonuclease activity.

* Corresponding author. Mailing address: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Phone: (617) 432-1864. Fax: (617) 432-3362. E-mail: ccr{at}hms.harvard.edu

{triangledown} Published ahead of print on 17 June 2009.

{dagger} Present address: Foley and Lardner LLP, 11250 El Camino Real, Suite 200, San Diego, CA 92130.

Journal of Virology, September 2009, p. 8418-8427, Vol. 83, No. 17
0022-538X/09/$08.00+0     doi:10.1128/JVI.00855-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»