Nucleic Acids Research Advance Access originally published online on February 11, 2009
Nucleic Acids Research 2009 37(6):2037-2052; doi:10.1093/nar/gkp064
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Nucleic Acids Research, 2009, Vol. 37, No. 6 2037-2052
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Characterization of DNA polymerase X from Thermus thermophilus HB8 reveals the POLXc and PHP domains are both required for 3'–5' exonuclease activity
1Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, 2RIKEN SPring-8 Center, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 and 3Systems and Structural Biology Center, Yokohama Institute, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan
*To whom correspondence should be addressed. Tel: +81 6 6850 5434; Fax: +81 6 6850 5442; Email: rmasui{at}bio.sci.osaka-u.ac.jp
Received November 4, 2008. Revised January 23, 2009. Accepted January 23, 2009.
The X-family DNA polymerases (PolXs) comprise a highly conserved DNA polymerase family found in all kingdoms. Mammalian PolXs are known to be involved in several DNA-processing pathways including repair, but the cellular functions of bacterial PolXs are less known. Many bacterial PolXs have a polymerase and histidinol phosphatase (PHP) domain at their C-termini in addition to a PolX core (POLXc) domain, and possess 3'–5' exonuclease activity. Although both domains are highly conserved in bacteria, their molecular functions, especially for a PHP domain, are unknown. We found Thermus thermophilus HB8 PolX (ttPolX) has Mg2+/Mn2+-dependent DNA/RNA polymerase, Mn2+-dependent 3'–5' exonuclease and DNA-binding activities. We identified the domains of ttPolX by limited proteolysis and characterized their biochemical activities. The POLXc domain was responsible for the polymerase and DNA-binding activities but exonuclease activity was not detected for either domain. However, the POLXc and PHP domains interacted with each other and a mixture of the two domains had Mn2+-dependent 3'–5' exonuclease activity. Moreover, site-directed mutagenesis revealed catalytically important residues in the PHP domain for the 3'–5' exonuclease activity. Our findings provide a molecular insight into the functional domain organization of bacterial PolXs, especially the requirement of the PHP domain for 3'–5' exonuclease activity.