A C-terminal Myb Extension Domain Defines a Novel Family of Double-strand Telomeric DNA-binding Proteins in Arabidopsis

Abstract

Little is known about the protein composition of plant telomeres. We queried the Arabidopsis thaliana genome data base in search of genes with similarity to the human telomere proteins hTRF1 and hTRF2. hTRF1/ hTRF2 are distinguished by the presence of a single Myb-like domain in their C terminus that is required for telomeric DNA binding in vitro. Twelve Arabidopsis genes fitting this criterion, dubbed TRF-like (TRFL), fell into two distinct gene families. Notably, TRFL family 1 possessed a highly conserved region C-terminal to the Myb domain called Myb-extension (Myb-ext) that is absent in TRFL family 2 and hTRF1/hTRF2. Immunoprecipitation experiments revealed that recombinant proteins from TRFL family 1, but not those from family 2, formed homodimers and heterodimers in vitro. DNA binding studies with isolated C-terminal fragments from TRFL family 1 proteins, but not family 2, showed specific binding to double-stranded plant telomeric DNA in vitro. Removal of the Myb-ext domain from TRFL1, a family 1 member, abolished DNA binding. However, when the Myb-ext domain was introduced into the corresponding region in TRFL3, a family 2 member, telomeric DNA binding was observed. Thus, Myb-ext is required for binding plant telomeric DNA and defines a novel class of proteins in Arabidopsis.Telomeres are the specialized nucleoprotein structures that comprise the natural ends of linear eukaryotic chromosomes and ensure their complete replication and stability (1, 2). In most eukaryotes, telomeric DNA is composed of tandem arrays of simple G-rich repeat sequences terminating in a singlestrand 3Ј-overhang, which is maintained through the action of the telomerase reverse transcriptase (1). Both the double and single-strand regions of the telomere are coated with nonhistone proteins that provide protection for telomeric DNA and regulate telomerase access to the chromosome terminus. Proteins that bind double-strand telomeric DNA are typified in vertebrates by TRF1 and TRF2 and in budding and fission yeast by Rap1 and Taz1,.Human TRF1 (hTRF1) 1 behaves as a negative regulator of telomere length; overexpression results in telomere shortening, whereas a dominant negative allele induces telomere elongation (9, 10). hTRF1 mediates telomere length control through interactions with other telomere-associated factors including tankyrase (11), TIN2 (12), PinX1 (13), and Pot1 (14). Although hTRF2 contributes to telomere length regulation (10), its major function is to conceal telomere ends from detection as doublestrand breaks (15, 16). Inhibition of hTRF2 in cultured human cells results in the loss of the 3Ј-overhang and the formation of covalently fused telomeres (15). In addition, compromised hTRF2 function culminates in cell cycle arrest and ATM/p53-mediated apoptosis (16).The functional domains of vertebrate TRF1 and TRF2 have been studied in some detail (17). The two proteins have similar molecular masses (50 -60 kDa), and resemble each other in domain structure. Although the N terminus is highly acidic in hTRF1 and highly basic in...

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

“…Although the exact composition of the complex varies between species, vertebrates, yeasts, plants, and ciliates all use a series of structurally related proteins to protect their telomeres. The G-overhang binding proteins all bind Gstrand DNA through a conserved OB fold motif, while the double-stranded DNA-binding proteins bind via a conserved myb motif (23,37,46).In vertebrate cells, telomeres are packaged by a core complex of six proteins which function both in telomere protection and in telomere length regulation (10). This core complex (which is sometimes called shelterin) contains two doublestranded DNA-binding proteins, TRF1 and TRF2, which anchor the complex along the length of the telomere, and the TRF1/2 interaction partners Rap1, TIN2, and TPP1.…”

An N-terminal region of a Myb-like protein is involved in its intracellular localization and activation of a gibberellin-inducible proteinase gene in germinated rice seeds

“…C-terminal Myb domain proteins are conserved in plants and are structurally related to human telomeric repeat binding factors. These Myb proteins make up the telomeric repeat binding factor-like (TRFL) family, 28) which is divided into 2 types. CTMyb1 belongs to the type II group (Supplemental Fig.…”

“…1(B)). 27) A phylogenetic analysis of the sequences of the C-terminal Myb domain proteins of Arabidopsis thaliana, 28) rice (AK071645 and AK242392), 29,30) and Catharanthus roseus 25) revealed that this protein family is classified into 2 major groups: I and II (Supplemental Fig. 2).…”

“…Type I includes AtTRFL1 and AtTRP1 of A. thaliana, which have been shown to bind specifically to plant telomeric DNA. 28) Rice RTBP1 is categorized as type I and also binds telomeric DNA. 29) These characteristics indicate that the type I proteins are telomeric DNAbinding proteins.…”

Roles of NtGTBP1 in telomere stability

“…1 [3][4][5] However, situation of singlestranded telomeric binding proteins is complicated. Pot1, a well-known shelterin complex protein, has single-stranded telomere repeat binding activity in yeasts and mammals but no DNA binding activity in Arabidopsis, despite the fact that it is necessary for the proper maintenance of telomere integrity.…”