Structure of the DNA-binding domain of NgTRF1 reveals unique features of plant telomere-binding proteins

Sunggeon Ko 0 2 y Sung-Hoon Jun 1 y Hansol Bae 1 Jung-Sue Byun 0 1 Woong Han 0 2 Heeyoung Park 0 2 Seong Wook Yang 1 Sam-Yong Park 4 Young Ho Jeon 3 Chaejoon Cheong 3 Woo Taek Kim 1 Weontae Lee 0 2 Hyun-Soo Cho 0 1 0 Protein Network Research Center, College of Life Sciences and Biotechnology, Yonsei University , Seoul 120-749, Korea 1 Department of Biology 2 Department of Biochemistry 3 Magnetic Resonance Team, Korea Basic Science Institute (KBSI) , Ochang, Chungbuk 363-883, Korea 4 Protein Design Laboratory, Yokohama City University , Suehiro 1-7-29, Tsurumi-ku, Yokohama 230-0045, Japan Telomeres are protein-DNA elements that are located at the ends of linear eukaryotic chromosomes. In concert with various telomere-binding proteins, they play an essential role in genome stability. We determined the structure of the DNAbinding domain of NgTRF1, a double-stranded telomere-binding protein of tobacco, using multidimensional NMR spectroscopy and X-ray crystallography. The DNA-binding domain of NgTRF1 contained the Myb-like domain and C-terminal Myb-extension that is characteristic of plant double-stranded telomere-binding proteins. It encompassed amino acids 561-681 (NgTRF1561-681), and was composed of 4 a-helices. We also determined the structure of NgTRF1561-681 bound to plant telomeric DNA. We identified several amino acid residues that interacted directly with DNA, and confirmed their role in the binding of NgTRF1 to telomere using site-directed mutagenesis. Based on a structural comparison of the DNA-binding domains of NgTRF1 and human TRF1 (hTRF1), NgTRF1 has both common and unique DNA-binding properties. Interaction of Myb-like domain with telomeric sequences is almost identical in NgTRF1561-681 with the DNA-binding domain of hTRF1. The interaction of Arg-638 with the telomeric DNA, which is unique in NgTRF1561-681, may provide the structural explanation for the specificity of NgTRF1 to the plant telomere sequences, (TTTAGGG)n. - INTRODUCTION Telomeres are essential for eukaryotic genome stability (1). During the last decade, telomeres have been the subject of intense study because of the link between telomere function and cancer and aging (2,3). Telomeric DNA consists of tandem repeats of simple conserved sequences, and functions in maintaining the integrity of flanking chromosomal sequences during replication (1,4). Telomeric DNA that is shortened during replication is restored through the action of telomerase, a reverse-transcriptase that synthesizes telomeric DNA using its own RNA molecule as a template (5,6). The synthesis of telomeres by telomerase and telomere length is regulated by numerous telomere-binding proteins. While the function of telomere-binding proteins in the regulation of telomere length is well characterized (7), other functions of them have also been described. The telomere-binding protein complex enables the DNA repair machinery to distinguish telomere ends from doublestranded DNA breaks. Defects in the telomere-binding protein complex trigger DNA damage response pathways that arrest the cell cycle and activate cell senescence or apoptosis (810). Telomere-binding proteins also protect telomeres from inappropriate DNA repair reactions, such as end-to-end joining and exonucleolytic digestion (11). In humans, six telomere-specific proteins have been known to form a complex (12). Of them, three proteins, hTRF1, hTRF2 and hPOT1, directly bind to telomeric DNA sequences and they are interconnected by two additional proteins, hTIN2 and hTPP1. hTRF1 and hTRF2 are double-stranded DNA-binding proteins, while hPOT1 binds to single-stranded DNA. hTRF1 forms homodimers, and possesses a Myb-like domain through which it binds to specific DNA sequences (1315). The role of hTRF1 in the regulation of telomere length has been demonstrated by gain-of-function studies (7,15) in which overexpression of wild-type allele caused telomere length to shorten and expression of a dominant negative allele resulted in progressive elongation of telomeres, until a new equilibrium was achieved. hTRF2 is a paralog of hTRF1 and its primary function is in telomere capping, which prevents end-to-end joining (11,16,17). hPOT1 has been proposed to function downstream of hTRF1 to relay the negative regulation to the telomere terminus (18). Several telomeric proteins have been identified in yeast. In the budding yeast Saccharomyces cerevisiae, RAP1 (scRAP1) is a doublestranded DNA-binding protein, and the primary telomere-binding protein (1921). Excess scRAP1 bound at the telomere negatively regulates telomere elongation in cis through the inhibition of telomerase activity (22,23). However, while scRAP1 is functionally analogous to hTRF1, the two proteins are not homologous. In contrast, fission yeast contains an ortholog of hTRF1, TAZ1, which binds to telomeric DNA duplexes and negatively regulates telomere length (24). The biological functions of telomeres and telomerebinding proteins have been studied extensively in humans and yeast. Double- or single-stranded telomere-binding proteins in plants have also been identified, which indicates that this class of proteins has been conserved throughout evolution (2528). In Arabidopsis, there are at least 12 TRF-like (TRFL) genes that have a single Myb-like domain in their C-terminal region and they fall into two distinct gene families based on the presence or absence of the C-terminal Myb-extension (29). Recombinant TRFL family 1 proteins, which contain C-terminal Myb-extension form homo- and hetero-dimers and specifically interact with plant double-stranded telomeric DNA in vitro. TRFL family 2 proteins lack the C-terminal Myb-extension, similarly to nonplant telomere-binding proteins such as hTRF1 and hTRF2, but they cannot bind to telomeric DNA. Single myb histone (SMH) family proteins, which have a single Myb-like domain in their N-terminal region, also bind telomere DNA repeats in vitro and they are plant specific (30,31). The protein AtTRB1, a member of SMH family, interacts with the Arabidopsis homolog protein of hPOT1, AtPot1, suggesting its plant telomere-specific role (32). The physiological functions of telomere-binding proteins in plant have been studied recently. The expression of NgTRF1, a tobacco double-stranded telomere-binding protein, is regulated tightly in correlation with cell division and the cell cycle (27). Overexpression of NgTRF1 resulted in a shorter telomere length compared to wild-type plants, whereas decreased expression of NgTRF1 resulted in a longer telomere length (33). Moreover, these perturbations of the expression of NgTRF1 caused apoptotic cell death. Recently, the in vivo function of rice telomere-binding protein, RTBP1, has been studied at the plant level (34). Loss-offunction (amorphic or hypomorphic) mutants of RTBP1 exhibited defects in both vegetative and reproductive development, and these phenotypes correlated with the gradual acquisition of dysfunctional telomeres. The structures of d (...truncated)