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

J. Biol. Chem., Vol. 279, Issue 39, 40405-40411, September 24, 2004

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 279/39/40405    most recent M401752200v1 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 Chen, L. S. Articles by Sheppard, T. L. Search for Related Content PubMed PubMed Citation Articles by Chen, L. S. Articles by Sheppard, T. L. Social Bookmarking                      What's this?

Chain Termination and Inhibition of Saccharomyces cerevisiae Poly(A) Polymerase by C-8-modified ATP Analogs^*

Lisa S. Chen and Terry L. Sheppard

From the Department of Chemistry and the Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, Illinois 60208-3113

The nucleotide substrate specificity of yeast poly(A) polymerase (yPAP) toward various C-2- and C-8-modified ATP analogs was examined. ³²P-Radiolabeled RNA oligonucleotide primers were incubated with yPAP in the absence of ATP to assay polyadenylation using unnatural ATP substrates. The C-2-modified ATP analogs 2-amino-ATP and 2-chloro (Cl)-ATP were excellent substrates for yPAP. 8-Amino-ATP, 8-azido-ATP, and 8-aza-ATP all produced chain termination of polyadenylation, and no primer extension was observed with the C-8-halogenated derivatives 8-Br-ATP and 8-Cl-ATP. The effects of modified ATP analogs on ATP-dependent poly(A) tail synthesis by yPAP were also examined. Whereas C-2 substitution (2-amino-ATP and 2-Cl-ATP) had little effect on poly(A) tail length, C-8 substitution produced moderate (8-amino-ATP, 8-azido-ATP, and 8-aza-ATP) to substantial (8-Br-ATP and 8-Cl-ATP) reduction in poly(A) tail length. To model the biochemical consequences of 8-Cl-Ado incorporation into RNA primers, a synthetic RNA primer containing a 3'-terminal 8-Cl-AMP residue was prepared. Polyadenylation of this modified RNA primer by yPAP in the presence of ATP was blocked completely. To probe potential mechanisms of inhibition, two-dimensional NMR spectroscopy experiments were used to examine the conformation of two C-8-modified AMP nucleotides, 8-Cl-AMP and 8-amino-AMP. C-8 substitution in adenosine analogs shifted the ribose sugar pucker equilibrium to favor the DNA-like C-2'-endo form over the C-3'-endo (RNA-like) conformation, which suggests a potential mechanism for polyadenylation inhibition and chain termination. Base-modified ATP analogs may exert their biological effects through polyadenylation inhibition and thus may provide useful tools for investigating polyadenylation biochemistry within cells.

Received for publication, February 17, 2004 , and in revised form, June 22, 2004.

^* This work was supported by National Institutes of Health Grant RO1 CA85915-02 and Leukemia and Lymphoma Society Grant 6505-00. The MALDI-TOF mass spectrometer was purchased with funds provided by National Institutes of Health Scientific Instrumentation Grant 1-S10-RR13810. The Center for Structural Biology at Northwestern University was supported by the Robert H. Lurie Comprehensive Cancer Center. 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.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. S1–S3 and Table S1.

To whom correspondence should be addressed: Dept. of Chemistry, Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208-3113. Tel.: 847-467-7636; Fax: 847-491-7713; E-mail: t-sheppard{at}northwestern.edu.

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

This article has been cited by other articles:

				C. M. Stellrecht, C. J. Phillip, F. Cervantes-Gomez, and V. Gandhi Multiple Myeloma Cell Killing by Depletion of the MET Receptor Tyrosine Kinase Cancer Res., October 15, 2007; 67(20): 9913 - 9920. [Abstract] [Full Text] [PDF]

				K. Balakrishnan, W. G. Wierda, M. J. Keating, and V. Gandhi Mechanisms of Cell Death of Chronic Lymphocytic Leukemia Lymphocytes by RNA-Directed Agent, 8-NH2-Adenosine Clin. Cancer Res., September 15, 2005; 11(18): 6745 - 6752. [Abstract] [Full Text] [PDF]

				K. Balakrishnan, C. M. Stellrecht, D. Genini, M. Ayres, W. G. Wierda, M. J. Keating, L. M. Leoni, and V. Gandhi Cell death of bioenergetically compromised and transcriptionally challenged CLL lymphocytes by chlorinated ATP Blood, June 1, 2005; 105(11): 4455 - 4462. [Abstract] [Full Text] [PDF]