Telomeric Dna And Associated Protein Complexes Biology

The establishment of these functions rely on specialized telomeric DNA and associated protein complexes (Stewart et al., 2012). Telomeric DNA contains several hundreds to thousands of non-coding repetitive Guanidine-rich DNA sequences that are unique in different organisms (e.g. GGTTAG in vertebrates and GGTTAC(A)(C)G(0-6) in fission yeast), terminating a 3' G-overhang at the very end (Xu, 2011). Telomeric DNA is coated by numerous proteins to form different structural and functional complexes to regulate protection and replication of telomere (Palm and de Lange, 2008). Telomere-associated proteins can be divided into three groups according to their functions and localizations. The first one is telomerase complex, who can de novo extends telomeric DNA to the 3' ends of chromosomes to complete the genome replication (Blackburn and Collins, 2011). The second group is telomere-binding proteins. They specifically associate with telomere DNA to from a protective cap to shield telomere from inappropriate DNA repair (Palm and de Lange, 2008). They also collaborate to regulate telomerase activity to maintain telomere length (Sampathi and Chai, 2011; Stewart et al., 2012). The third group is telomere accessory factors, including proteins in DNA repair and damage signaling pathway and in protein degradation pathway (Palm and de Lange, 2008). These accessory factors have important roles in telomere biogenesis and maintenance, but they also have more general roles in other cellular processes (de Lange, 2005; Palm and de Lange, 2008; Perry et al., 2010). More often, they only transiently associate with telomere during cell cycle and much less abundant than telomere-specific proteins (Palm and de Lange, 2008). In this review, we will focus on proteins from the first and second group, which are telomere-specific.

Structural elucidation has been an important tool to infer the evolutionary relationship of telomere proteins across species (Gelinas et al., 2009; Sun et al., 2009; Wang et al., 2007). Hitherto high resolution structures of several telomere-associated proteins have been reported, which reveal some important features about the conservation and plasticity of these telomeric proteins (Lewis and Wuttke, 2012; Mason et al., 2011). Here, the recent progresses on structural studies on telomerase complex and telomere-binding protein complexes will be discussed to reveal some conserved structural elements with diversified functions. The organization and functions of telomeric complexes in three representing species will also be addressed to highlight the common theme and evolutionary plasticity of telomeric proteins involving in telomere homeostasis and protection.

1. Structural biology of Telomerase

Telomere can be extended by transposition, homology recombination and de novo synthesis (Eisenstein, 2011; Sampathi and Chai, 2011). The de novo synthesis of telomere is carried out by a special ribonucleoprotein complex, telomerase (Blackburn and Collins, 2011). It contains a reverse transcriptase protein component (TERT) and an internal RNA template (TR) to mediate nucleotide addition to the 3' end of G overhang (Blackburn and Collins, 2011; Podlevsky and Chen, 2012). Additional regulatory or structural proteins associate with the core TERT-TR complex to form a functional holoenzyme (Collins, 2011).

TERT proteins in most species contain four domains: a telomerase essential N-terminal (TEN) domain, a telomerase RNA binding (TRBD )domain, a reverse transcriptase (RT) domain, and a C-terminal extension (CTE) domain (Mason et al., 2011). The TEN domain interacts with both telomeric ssDNA and TR RNA component, and can promote processive repeat synthesis by telomerase (Jacobs et al., 2006). TRBD domain assure specific recognition between TERT and TR (Rouda and Skordalakes, 2007). RT domain is similar to retrotransposon RTs, and is the active site for catalysis of dNTP addition (Gillis et al., 2008). The CTE domain may enhance the DNA association, but the exact role is not clear (Hossain et al., 2002). The domain organization of TERT is quite conserved, as the variation often happens at the connecting loops between these domains (Lewis and Wuttke, 2012; Mason et al., 2011). In contrast, the RNA component of telomerase vary a lot, with disparate sequences and diversified sizes from about 150 nt in ciliates to 1300 nt in budding yeasts (Theimer and Feigon, 2006). Although conserved core secondary structure elements of TR were identified (Chen and Greider, 2004), the precise roles of these elements are still in debate.

Up to date, some individual telomerase domains and RNA fragments have been structural characterized (Lewis and Wuttke, 2012; Theimer and Feigon, 2006) (Table 1). However, the high-resolution structures of telomerase holoenzymes are still missing. Here, we will focus on structural studies of Tetrahymena thermophila telomerase, since this is the first identified and best characterized telomerase (Greider and Blackburn, 1985, 1989; Lingner et al., 1997). Besides TERT and TR, T. thermophila telomerase holoenzyme contain four regulatory proteins, p20, p45, p66 and p75 (Collins, 2011). TERT, TR and p65 comprise a stable catalytic core complex; p75, p45, and p19 comprise a telomere adaptor subcomplex (TASC) (Collins, 2011) (Figure 1A). Another telomerase-associated factor, Teb1, collaborates with TASC to stimulate the repeat addition processivity (RAP) of telomerase (Zeng et al., 2010). Several domains of T. thermophila TERT, including TtTEN (Jacobs et al., 2006), TtTRBD (Rouda and Skordalakes, 2007) and a homologous RT domain from Tribolium castaneum (Gillis et al., 2008), have high-resolution structures (Figure 1B). The structures of two important structural elements (Stem II and Stem IV) in T. thermophila TR have been determined (Chen et al., 2006; Richards et al., 2006a; Richards et al., 2006b). The complex structures of some telomerase-regulatory proteins, such as P65 and Teb1 in complex with RNA and ssDNA respectively, have also been reported (Singh et al., 2012; Zeng et al., 2011) (Figure 1B). These structures reveal important information about the assembly and activity regulation of telomerase.

The structure of TtTEN domain presents a novel protein fold (Jacobs et al., 2006). Mutagenesis of some conserved residues in a groove on its surface shows these residues are crucial for binding of telomeric ssDNA and promote telomerase activity. A flexible C-terminal loop of TEN domain is positively charged and is involved in RNA binding. The multiple roles of TEN domains highlight the importance of this domain in telomerase catalysis activity.

TtTRBD domain is a kinked structure with two asymmetric helical lobes (Rouda and Skordalakes, 2007), which is ideal for TR binding. Two conserved motifs (CP and T motifs) important for RNA binding, are localized at the center hinge region of TRBD domain, forming the potential RNA-binding pocket. The structure of isolated TtTRBD domain is almost identical to TRBD structure in full length TcTERT (T. castenem TERT), suggesting T.thermophila TERT may adopt similar structure as T. castenem TERT structure.

TcTERT structure shows a striking ring-like structure composed of three domains: TRBD, RT and CTE. RT domains processes the canonical RT motifs 1, 2, A, B', C, D, and E, organized in a right-hand-like structure (Gillis et al., 2008). RT and CTE domains form a canonical palm-finger-thumb structure, and TRBD domain contacts with CTE domain to complete the ring. The complex structure of TcTERT with a model RNA-DNA hybrid confirms that the nucleic acid sits in the center of the ring with significant contacts with fingers, palm and thumb domains (Mitchell et al., 2010a). The active site is in the palm of the RT and contains universally conserved catalytic aspartates (Asp251, Asp343 and Asp344). Contacts between protein and nucleic acid places 3′-end hydroxyl of the DNA primer at the active site of the enzyme for nucleotide addition, suggesting current structure is in an active telomerase conformation. However, since the TR component associated with TcTERT has not been identified, the real picture of telomerase action is still on hold until the elucidation of the structure TERT complex with an endogenous TR.

T. thermophila possesses the shortest TR with only 159-nt, but includes all the conserved motifs (Theimer and Feigon, 2006). It contains four helices, Stem I to IV, and several single-stranded regions with important functions (Egan and Collins, 2012) (Figure 1A). Stem I is involved in long-range base pairing and crucial for TERT binding (O'Connor et al., 2005). Stem II contains template boundary element (TBE) for 5′ template boundary definition, and also interacts with TRBD of TERT (Cunningham and Collins, 2005). Stem IIIa and IIIb contribute to form RNA pseudoknot involved in telomerase assembly and activity (Cunningham and Collins, 2005; Gilley and Blackburn, 1999; Lai et al., 2003). The 9-nt template sequence is localized between stem II and III. Stem IV is involved in nucleotide addition processivity and interaction with TEN domain of TERT (Robart et al., 2010). The solution structure of 23-nt T. thermophila stem II reveals that  stem II forms a well-defined helix with two unpaired adenines and a pentaloop (Richards et al., 2006a), in sharp contrast with previous biochemical data that stem II alone is unstructured (Sperger and Cech, 2001). Comparison of same TBE loop in stem II structure of Haloarcula marismortui  23s rRNA indicates that similar RNA-protein interactions may occur in TERT/TR complex. The NMR structure of 43-nt T.thermophila stem IV was obtained from two overlapping fragments corresponding to the template-proximal and distal parts of the stem IV. The complete stem IV structure exhibits a severely kinked structure, with a rigidly defined distal loop linked to a conformational flexible template-proximal region (Chen et al., 2006; Richards et al., 2006b). The GA bulge in the middle of stem-loop IV sharply kinks the entire structure. The nucleotides contributing to formation of such a specific structure are well conserved, and mutations of these nucleotides disrupt the enzyme activity while no effect on TERT binding, suggesting a complicated mechanism for reposition of stem IV during catalytic cycle.

The catalytic core of T.thermophila telomerase is composed of TR, TERT and an essential La family protein P65. P65 is important for TR accumulation and holoenzyme assembly. P65 is a member of LARP7 family proteins, and contains four domains: an N-terminal domain, a La motif (LAM), an RNA recognition motif (RRM), a C-terminal domain. The C-terminal domain is an atypic RRM domain as shown by NMR structure (Singh et al., 2012). Structure of P65 C-terminal domain with stem IV of TR revealed that specific protein-RNA recognition induce significant conformational change on both protein and RNA. The previously disordered C-terminal extension in unliganded P65 protein converts to an α helix in the complex, which is necessary for hierarchical assembly of TERT with p65-TR (Singh et al., 2012). The protein-binding also induce bend in stem IV of TR, possibly positioning it for interaction with TRBD domain (Singh et al., 2012).

Teb1 association with the T.thermophila telomerase catalytic core can convert the limited repeat addition processivity (RAP) of the catalytic core to the high RAP of endogenously assembled holoenzyme. Teb1 contains three putative OB (Oligonucleotide or oligosaccharide-Binding) folds, which were confirmed by crystal structures of these domains (Zeng et al., 2011). Two N-terminal OB folds (Teb1OB1+2) achieve high affinity and selectivity of telomeric single-stranded DNA (ssDNA) recognition by specific protein structures. The C-terminal OB fold (Teb1OB3) only marginally contribute to DNA binding, but definitely crucial for high RAP activity (Zeng et al., 2011). These results suggest a model that the initial recruitment of telomerase to telomeric ssDNA tracts involves Teb1OB1,2 recognition of telomeric repeats. Subsequent capture of the 3′ end by the active site of the telomere catalytic core could then favor Teb1OB3-ssDNA contact, trapping product in a sliding-clamp-like manner that does not require high-affinity DNA binding for high stability of enzyme-product association (Zeng et al., 2011).

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