STRUCTURAL AND FUNCTIONAL ANALYSIS OF DUPLEX TELOMERIC DNA-BINDING PROTEINS IN ARABIDOPSIS
Telomere, the nucleoprotein complex, locates at the end of linear chromosome and is essential for the maintenance of the integrity of genome in eukaryotic cell.
One gene encoding two distinct duplex telomeric DNA-binding proteins, TRP4 and TRFL1, by alternative splicing in A. thaliana.
The telomeric DNA in most eukaryotic cells is composed of tandem array of duplex, short,
and GT-rich repeated sequences followed by an extension of the GT-rich strand of telomeric DNA in a direction from 5' to 3' toward the end of a chromosome. Synthesis of telomeric DNA is catalyzed by telomerase, however, studies in yeast and mammalian cells have shown that the access of telomerase to telomere and the activity of telomerase are controlled by various proteins associated with telomeric DNA including the duplex telomeric DNA-binding proteins. Previously we have identified a duplex telomeric DNA-binding protein AtTRP1 from Arabidopsis thaliana. The DNA-binding domain of AtTRP1 has been defined to the Cterminal 115 residues of this molecule. In the past few years, we have focused on the following area to understand the structure and function of AtTRP1.
1. Molecular cloning of Arabidopsis cDNAs encoding AtTRP1 homologs and biochemical characterization of the knockout mutants of genes encoding AtTRP1 and its homologs.
2. Determination of the structure of the DNAbinding domain of AtTRP1 by nuclear magnetic resonance (NMR) spectrum.
3. Biochemical characterization of AtTRP1 to identify additional domains.
4. Identification of proteins interacting with AtTRP1.
NMR study reveals that the DNA-binding domain of AtTRP1 utilizes four helices to recognize the telomeric DNA.
A gene family, termed AtTRP, was identified in A. thaliana to contain six members including AtTRP1 which encode at least seven distinct duplex telomeric DNAbinding proteins that are highly homologous one another in several regions including the C-terminal DNA-binding domain. Knockout of any single gene in AtTRP family did not result in a significant change in telomere length of the corresponding mutants, suggesting that these genes may be functionally redundant in the regulation of telomere length in Arabidopsis.
The DNA-binding domain of AtTRP1 contains four-helix tetrahedron instead of the three-helix bundle structure found in Myb motifs and other duplex telomeric DNAbinding proteins. AtTRP1 utilizes these four helices in the DNA-binding domain to recognize the telomeric repeats centered on the sequence GGTTTAG.
Biochemical studies have shown that AtTRP1 contains additional domains that may be important for the biological function of this protein and its homologs. Several proteins interacting with AtTRP1 have been isolated by biochemical methods and
sequencing of these proteins is under way.
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