HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 282, Issue 46, 33444-33451, November 16, 2007

This Article Full Text Full Text (PDF) All Versions of this Article: 282/46/33444    most recent M705826200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Weiner, B. E. Articles by Chazin, W. J. Search for Related Content PubMed PubMed Citation Articles by Weiner, B. E. Articles by Chazin, W. J. Social Bookmarking                      What's this?

An Iron-Sulfur Cluster in the C-terminal Domain of the p58 Subunit of Human DNA Primase^*

Brian E. Weiner, Hao Huang^¶, Brian M. Dattilo, Mark J. Nilges^||, Ellen Fanning^¶, and Walter J. Chazin^**1

From the Departments of Biochemistry, ^¶Biological Sciences, and ^**Chemistry, the Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232 and the ^||Illinois EPR Research Center, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

DNA primase synthesizes short RNA primers that are required to initiate DNA synthesis on the parental template strands during DNA replication. Eukaryotic primase contains two subunits, p48 and p58, and is normally tightly associated with DNA polymerase . Despite the fundamental importance of primase in DNA replication, structural data on eukaryotic DNA primase are lacking. The p48/p58 dimer was subjected to limited proteolysis, which produced two stable structural domains: one containing the bulk of p48 and the other corresponding to the C-terminal fragment of p58. These domains were identified by mass spectrometry and N-terminal sequencing. The C-terminal p58 domain (p58C) was expressed, purified, and characterized. CD and NMR spectroscopy experiments demonstrated that p58C forms a well folded structure. The protein has a distinctive brownish color, and evidence from inductively coupled plasma mass spectrometry, UV-visible spectrophotometry, and EPR spectroscopy revealed characteristics consistent with the presence of a [4Fe-4S] high potential iron protein cluster. Four putative cysteine ligands were identified using a multiple sequence alignment, and substitution of just one was sufficient to cause loss of the iron-sulfur cluster and a reduction in primase enzymatic activity relative to the wild-type protein. The discovery of an iron-sulfur cluster in DNA primase that contributes to enzymatic activity provides the first suggestion that the DNA replication machinery may have redox-sensitive activities. Our results offer new horizons in which to investigate the function of high potential [4Fe-4S] clusters in DNA-processing machinery.

Received for publication, July 16, 2007 , and in revised form, September 18, 2007.

Addendum—During the review of the manuscript, Klinge and colleagues (59) reported the presence of an iron-sulfur cluster in DNA primase from S. solfataricus and S. cerevisiae. Our findings are in agreement with their data and extend them by identifying the cluster in human DNA primase and by showing that the cluster is contained in a distinct and well folded structural domain within the p58 subunit.

^* This work was supported by National Institutes of Health Grants GM65484 (to W. J. C.) and GM52948 (to E. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Center for Structural Biology, Vanderbilt University, 465 21st Ave. S., Ste. 5140, Nashville, TN 37232-8725. Tel.: 615-936-2210; Fax: 615-936-2211; E-mail: walter.chazin{at}vanderbilt.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. T. P. Yeeles, R. Cammack, and M. S. Dillingham An Iron-Sulfur Cluster Is Essential for the Binding of Broken DNA by AddAB-type Helicase-Nucleases J. Biol. Chem., March 20, 2009; 284(12): 7746 - 7755. [Abstract] [Full Text] [PDF]

				B. Ren, X. Duan, and H. Ding Redox Control of the DNA Damage-inducible Protein DinG Helicase Activity via Its Iron-Sulfur Cluster J. Biol. Chem., February 20, 2009; 284(8): 4829 - 4835. [Abstract] [Full Text] [PDF]

				O. Stehling, D. J. A. Netz, B. Niggemeyer, R. Rosser, R. S. Eisenstein, H. Puccio, A. J. Pierik, and R. Lill Human Nbp35 Is Essential for both Cytosolic Iron-Sulfur Protein Assembly and Iron Homeostasis Mol. Cell. Biol., September 1, 2008; 28(17): 5517 - 5528. [Abstract] [Full Text] [PDF]