OB Fold-containing Protein 1 (OBFC1), a Human Homolog of Yeast Stn1, Associates with TPP1 and Is Implicated in Telomere Length Regulation

The telosome/shelterin, a six-protein complex formed by TRF1, TRF2, RAP1, TIN2, POT1, and TPP1, functions as the core of the telomere interactome, acting as the molecular platform for the assembly of higher order complexes and coordinating cross-talks between various protein subcomplexes. Within the telosome, there are two oligonucleotide- or oligosaccharide-binding (OB) fold-containing proteins, TPP1 and POT1. They can form heterodimers that bind to the telomeric single-stranded DNA, an activity that is central for telomere end capping and telomerase recruitment. Through proteomic analyses, we found that in addition to POT1, TPP1 can associate with another OB fold-containing protein, OBFC1/AAF44. The yeast homolog of OBFC1 is Stn1, which plays a critical role in telomere regulation. We show here that OBFC1/AAF44 can localize to telomeres in human cells and bind to telomeric single-stranded DNA in vitro. Furthermore, overexpression of an OBFC1 mutant resulted in elongated telomeres in human cells, implicating OBFC1/AAF4 in telomere length regulation. Taken together, our studies suggest that OBFC1/AAF44 represents a new player in the telomere interactome for telomere maintenance.Telomeres are specialized linear chromosome end structures, which are regulated and protected by networks of protein complexes (1–4). Telomere length, structure, and integrity are critical for the cells and the organism as a whole. Telomere dysregulation can lead to DNA damage response, cell cycle checkpoint, genome instability, and predisposition to cancer (5–9). Mammalian telomeres are composed of double-stranded (TTAGGG)_n repeats followed by 3′-single-stranded overhangs (10). In addition to the telomerase that directly mediates the addition of telomere repeats to the end of chromosomes (11, 12), a multitude of telomere-specific proteins have been identified that form the telosome/shelterin complex and participate in telomere maintenance (9, 13). The telosome in turn acts as the platform onto which higher order telomere regulatory complexes may be assembled into the telomere interactome (14). The telomere interactome has been proposed to integrate the complex and labyrinthine network of protein signaling pathways involved in DNA damage response, cell cycle checkpoint, and chromosomal end maintenance and protection for telomere homeostasis and genome stability.Of the six telomeric proteins (TRF1, TRF2, RAP1, TIN2, POT1, and TPP1) that make up the telosome, TRF1 and TRF2 have been shown to bind telomeric double-stranded DNA (15, 16), whereas the OB³ fold-containing protein POT1 exhibits high affinities for telomeric ssDNA in vitro (17, 18). Although the OB fold of TPP1 does not show appreciable ssDNA binding activity, heterodimerization of TPP1 and POT1 enhances the POT1 ssDNA binding (17, 18). More importantly, POT1 depends on TPP1 for telomere recruitment, and the POT1-TPP1 heterodimer functions in telomere end protection and telomerase recruitment. Notably, the OB fold of TPP1 is critical for the recruitment of the telomerase (18). Disruption of POT1-TPP1 interaction by dominant negative inhibition, RNA interference, or gene targeting could lead to dysregulation of telomere length as well DNA damage responses at the telomeres (18–21).In budding yeast, the homolog of mammalian POT1, Cdc13, has been shown to interact with two other OB fold-containing proteins, Stn1 and Ten1, to form a Cdc13-Stn1-Ten1 (CST) complex (22, 23). The CST complex participates in both telomere length control and telomere end capping (22, 23). The presence of multiple OB fold-containing proteins from yeast to human suggests a common theme for telomere ssDNA protection (4). Indeed, it has been proposed that the CST complex is structurally analogous to the replication factor A complex and may in fact function as a telomere-specific replication factor A complex (23). Notably, homologs of the CST complex have been found in other species such as Arabidopsis (24), further supporting the notion that multiple OB fold proteins may be involved in evolutionarily conserved mechanisms for telomere end protection and length regulation. It remains to be determined whether the CST complex exists in mammals.Although the circuitry of interactions among telosome components has been well documented and studied, how core telosome subunits such as TPP1 help to coordinate the cross-talks between telomere-specific signaling pathways and other cellular networks remains unclear. To this end, we carried out large scale immunoprecipitations and mass spectrometry analysis of the TPP1 protein complexes in mammalian cells. Through these studies, we identified OB fold-containing protein 1 (OBFC1) as a new TPP1-associated protein. OBFC1 is also known as α-accessory factor AAF44 (36). Sequence alignment analysis indicates that OBFC1 is a homolog of the yeast Stn1 protein (25). Further biochemical and cellular studies demonstrate the association of OBFC1 with TPP1 in live cells. Moreover, we showed that OBFC1 bound to telomeric ssDNA and localized to telomeres in mammalian cells. Dominant expression of an OBFC1 mutant led to telomere length dysregulation, indicating that OBFC1 is a novel telomere-associated OB fold protein functioning in telomere length regulation.     

Identification of the Determinant Conferring Permissive Substrate Usage in the Telomere Resolvase, ResT

Linear genome stability requires specialized telomere replication and protection mechanisms. A common solution to this problem in non-eukaryotes is the formation of hairpin telomeres by telomere resolvases (also known as protelomerases). These enzymes perform a two-step transesterification on replication intermediates to generate hairpin telomeres using an active site similar to that of tyrosine recombinases and type IB topoisomerases. Unlike phage telomere resolvases, the telomere resolvase from the Lyme disease pathogen Borrelia burgdorferi (ResT) is a permissive enzyme that resolves several types of telomere in vitro. However, the ResT region and residues mediating permissive substrate usage have not been identified. The relapsing fever Borrelia hermsii ResT exhibits a more restricted substrate usage pattern than B. burgdorferi ResT and cannot efficiently resolve a Type 2 telomere. In this study, we determined that all relapsing fever ResTs process Type 2 telomeres inefficiently. Using a library of chimeric and mutant B. hermsii/B. burgdorferi ResTs, we mapped the determinants in B. burgdorferi ResT conferring the ability to resolve multiple Type 2 telomeres. Type 2 telomere resolution was dependent on a single proline in the ResT catalytic region that was conserved in all Lyme disease but not relapsing fever ResTs and that is part of a 2-amino acid insertion absent from phage telomere resolvase sequences. The identification of a permissive substrate usage determinant explains the ability of B. burgdorferi ResT to process the 19 unique telomeres found in its segmented genome and will aid further studies on the structure and function of this essential enzyme.Replication and protection of telomeric DNA are required to ensure the genomic stability of all organisms with linear replicons. Until quite recently, it was assumed that linearity is a property confined to the replicons of eukaryotes and certain primarily eukaryotic viruses. However, a growing body of evidence indicates that linear DNA is also found in a broad range of bacteriophages (1–6) and in bacteria themselves (7–10), including the Borrelia species that cause Lyme disease and relapsing fever (11, 12). A common solution to the end replication and protection problem in non-eukaryotes is the covalent sealing of DNA ends in the form of hairpins (2, 4–6, 10, 11, 13–16). Hairpin DNA is not recognized as a double-strand break, and continuous synthesis of DNA around the hairpin loop abolishes the end replication problem. However, mother and daughter replicons are covalently linked at the junction of their telomeres following DNA replication; separation of the two replicons and formation of new hairpin telomeres require a DNA breakage and reunion process referred to as telomere resolution (17, 18).Resolution of the linear chromosome and plasmids in Borrelia species and of the linear plasmid prophages from Escherichia coli, Yersinia enterocolitica, and Klebsiella oxytoca is performed by telomere resolvases (also referred to as protelomerases) (5, 19–21). A growing number of candidate telomere resolvases have been identified in the genomes of eukaryotic viruses, phages, and bacteria (22, 23). Telomere resolvases are DNA cleavage and rejoining enzymes related to tyrosine recombinases and type 1B topoisomerases (19, 21, 22, 24, 25). Telomere resolvase catalyzes a two-step transesterification reaction in which staggered cuts are introduced 6 bp apart on either side of the axis of symmetry in the replicated telomere substrate (5, 19, 21, 24). Cleavage is accompanied by the formation of a 3′-phosphotyrosyl protein-DNA linkage. Subsequent nucleophilic attack on opposing strands by the free 5′-OH groups in the nicked substrate creates covalently closed hairpin telomeres. A recent crystal structure of the Klebsiella phage telomere resolvase (TelK) in complex with its substrate identified the residues involved in catalysis (25); all but one of these residues are conserved in all telomere resolvases (22), implying that the basic catalytic mechanism underlying telomere resolution is conserved. However, telomere resolvase sequences vary substantially outside of the central catalytic region (25, 26), and the enzymes characterized to date demonstrate important differences in substrate usage that likely reflect functionally distinct mechanisms of substrate interaction.The Borrelia burgdorferi telomere resolvase, ResT, appears to be particularly divergent. It is substantially smaller than phage telomere resolvases, and unlike its phage counterparts (5, 20, 21), it cannot efficiently resolve negatively supercoiled DNA (19, 27), presumably reflecting differences in the substrates resolved by phage and Borrelia telomere resolvases in vivo. On the other hand, B. burgdorferi ResT can fuse hairpin telomeres in a reversal of the resolution reaction (28), a function that is not shared with the phage telomere resolvase TelK (25). It can also synapse replicated telomeres and catalyze the formation of Holliday junctions (29). The ability of ResT to promote hairpin fusion has been proposed as the mechanism underlying the ongoing genetic rearrangements that are a prominent feature of the B. burgdorferi genome (18, 28). Finally, B. burgdorferi ResT can tolerate a surprising amount of variation in its substrate (30, 31), a feature that is not shared by phage telomere resolvases (21). Although B. burgdorferi ResT appears to be more permissive with a greater scope of activities than other telomere resolvases, the sequences mediating most of its unique properties have not yet been identified.The B. burgdorferi genome contains a total of 19 distinct hairpin sequences, all of which must be resolved by ResT (31). These sequences can be classified into three groups based on the presence and positioning of the box 1 motif, which is a critical determinant of activity in phage and Borrelia telomere resolvases (see Fig. 1A) (21, 24, 30). A box 1-like motif is also found in many of the hairpin telomeres sequenced to date (6, 14, 32–35), although its function in telomere resolution is unknown. The box 1 consensus sequence (TAT(a/t)AT) closely resembles the −10/Pribnow box and TATA box consensus sequences of prokaryotic and eukaryotic promoters (TATAAT and TATA(a/t)A(a/t), respectively), which undergo transient deformations that predispose them to melting (36) and are intrinsically bent and anisotropically flexible (37). Therefore, box 1 may facilitate nucleation of hairpin folding and/or may confer an intrinsic bend or flexibility to substrates that is important for the resolution reaction.Open in a separate windowFIGURE 1.Species-specific resolution of Type I and 2 telomeres. A, a schematic showing the three types of hairpin telomere found on the linear replicons of the B. burgdorferi genome (see Ref. 31). The box 1 sequence in Type 1 and 2 telomeres is situated 1 and 4 nucleotides away from the axis of symmetry, respectively, whereas Type 3 telomeres contain no clear box 1. B, a schematic illustrating the telomere resolution reaction substrate and products is shown along with two ethidium bromide-stained agarose gels showing telomere resolution assays. The gels show resolution kinetics for B. burgdorferi and B. hermsii ResT on Type 1 and 2 telomeres (plasmid substrates pYT1/lp17L and pYT92/chromL, respectively).B. burgdorferi ResT can resolve telomeres in which box 1 is located at positions 1 and 4 nucleotides away from the axis of symmetry (Type 1 and 2 telomeres, respectively), as well as AT-rich telomeres without a box 1 sequence (Type 3 telomeres) (see Fig. 1A) (30, 31). B. burgdorferi ResT cleaves telomeres at a fixed position relative to the axis of symmetry, independent of the location of box 1 (30). Positioning of the enzyme for cleavage in all telomere types is most likely driven by sequence-specific interactions between ResT domains 2 (catalytic) and/or 3 (C-terminal) and a fixed element upstream of box 1 that is positioned 14 nucleotides from the axis of symmetry in all Borrelia telomeres (box 3 and adjacent nucleotides) (see Figs. 1A and ​and2)2) (26, 30, 31). In contrast, box 1 and axis-flanking nucleotides are not involved in high affinity and/or sequence-specific interactions with ResT and require the ResT N-terminal domain for full protection in DNase footprinting assays (26, 27). The most likely candidate for interactions with box 1 and axis-flanking nucleotides is a Borrelia-specific hairpin-binding region in the N terminus, which is thought to promote a pre-hairpinning step involving strand opening at the axis (38).Open in a separate windowFIGURE 2.Alignment of 11 Borrelia ResT sequences. Shown is ClustalW2 alignment of ResT amino acid sequences from five Lyme disease Borrelia species (B. afzelii, B. spielmanii, B. valaisiana, B. garinii, and B. burgdorferi), five relapsing fever Borrelia species (B. turicatae, B. parkeri, B. hermsii, B. recurrentis, and B. duttonii), and one avian Borrelia species (B. anserina) (generated using ClustalW2 from the EBI web site) (19, 39–42, 48, 49). The sequences for B. anserina, B. parkeri, and B. turicatae ResTs are reported for the first time in this study (respective GenBank accession numbers are {"type":"entrez-nucleotide","attrs":{"text":"FJ882620","term_id":"242263541","term_text":"FJ882620"}}FJ882620, {"type":"entrez-nucleotide","attrs":{"text":"FJ882621","term_id":"242263543","term_text":"FJ882621"}}FJ882621, and {"type":"entrez-nucleotide","attrs":{"text":"FJ882623","term_id":"242263547","term_text":"FJ882623"}}FJ882623). Sequences are arranged in order of similarity to neighboring sequences and are colored in JalView using the Zappo coloring scheme for identifying amino acids with similar physicochemical properties (50). Only residues that are identical in 100% of ResTs are indicated by colored shading. Arrows above the alignment indicate ResT domain boundaries identified by chymotrypsin digest, sequence comparison with other proteins, and HHsenser predictions (26, 51). The hairpin-binding motif found in cut-and-paste transposases is indicated beneath the alignment by white text on a black background (38). The positions corresponding to the active site residues in tyrosine recombinases, type IB topoisomerases, and TelK are indicated by blue asterisks below the sequence, with the active site tyrosine nucleophile at position 335 marked by a red asterisk (22, 25). The ringed black dot below position 326 indicates an amino acid in the active site region that differs in Lyme disease and relapsing fever ResTs. Sequences above the black line drawn between B. burgdorferi and B. turicatae are from Lyme disease Borrelia species; sequences below the black line are from relapsing fever Borrelia species. The ResT sequence from the avian Borrelia species B. anserina is shown at bottom.ResT from the relapsing fever Borrelia species Borrelia hermsii exhibits a more restricted substrate usage pattern in vitro when compared with ResT from the Lyme disease pathogen B. burgdorferi (39). Specifically, B. hermsii ResT is unable to efficiently resolve a Type 2 telomere. Therefore, B. burgdorferi ResT appears to be a more permissive enzyme than its relapsing fever counterpart. In this study, we investigated the basis for permissive substrate usage by B. burgdorferi ResT. Using a library of chimeric B. hermsii/B. burgdorferi ResTs, we mapped the sequence determinants in B. burgdorferi ResT that confer the ability to resolve multiple Type 2 telomeres. Surprisingly, this approach indicated that Type 2 telomere resolution was crucially regulated by a single proline residue located in a small Borrelia-specific insertion in the central catalytic region of ResT. The proline at this position was conserved in the ResTs from all Lyme disease Borrelia species but in none of the ResTs from relapsing fever Borrelia species, which were unable to efficiently resolve Type 2 telomeres in vitro. This study has identified a specific residue in ResT responsible for permissive substrate usage patterns.     

Splitting the BLOSUM Score into Numbers of Biological Significance

Mathematical tools developed in the context of Shannon information theory were used to analyze the meaning of the BLOSUM score, which was split into three components termed as the BLOSUM spectrum (or BLOSpectrum). These relate respectively to the sequence convergence (the stochastic similarity of the two protein sequences), to the background frequency divergence (typicality of the amino acid probability distribution in each sequence), and to the target frequency divergence (compliance of the amino acid variations between the two sequences to the protein model implicit in the BLOCKS database). This treatment sharpens the protein sequence comparison, providing a rationale for the biological significance of the obtained score, and helps to identify weakly related sequences. Moreover, the BLOSpectrum can guide the choice of the most appropriate scoring matrix, tailoring it to the evolutionary divergence associated with the two sequences, or indicate if a compositionally adjusted matrix could perform better.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29]     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Sgt1 Dimerization Is Negatively Regulated by Protein Kinase CK2-mediated Phosphorylation at Ser361

The kinetochore, which consists of centromere DNA and structural proteins, is essential for proper chromosome segregation in eukaryotes. In budding yeast, Sgt1 and Hsp90 are required for the binding of Skp1 to Ctf13 (a component of the core kinetochore complex CBF3) and therefore for the assembly of CBF3. We have previously shown that Sgt1 dimerization is important for this kinetochore assembly mechanism. In this study, we report that protein kinase CK2 phosphorylates Ser³⁶¹ on Sgt1, and this phosphorylation inhibits Sgt1 dimerization.The kinetochore is a structural protein complex located in the centromeric region of the chromosome coupled to spindle microtubules (1, 2). The kinetochore generates a signal to arrest cells during mitosis when it is not properly attached to microtubules, thereby preventing chromosome missegregation, which can lead to aneuploidy (3, 4). The molecular structure of the kinetochore complex of the budding yeast Saccharomyces cerevisiae has been well characterized; it is composed of more than 70 proteins, many of which are conserved in mammals (2).The centromere DNA in the budding yeast is a 125-bp region that contains three conserved regions, CDEI, CDEII, and CDEIII (5, 6). CDEIII (25 bp) is essential for centromere function (7) and is bound to a key component of the centromere, the CBF3 complex. The CBF3 complex contains four proteins, Ndc10, Cep3, Ctf13 (8–15), and Skp1 (14, 15), all essential for viability. Mutations in any of the CBF3 proteins abolish the ability of CDEIII to bind to CBF3 (16, 17). All of the kinetochore proteins, except the CDEI-binding Cbf1 (18–20), localize to the kinetochores in a CBF3-dependent manner (2). Thus, CBF3 is a fundamental kinetochore complex, and its mechanism of assembly is of great interest.We have previously found that Sgt1 and Skp1 activate Ctf13; thus, they are required for assembly of the CBF3 complex (21). The molecular chaperone Hsp90 is also required to form the active Ctf13-Skp1 complex (22). Sgt1 has two highly conserved motifs that are required for protein-protein interaction: the tetratricopeptide repeat (21) and the CHORD protein and Sgt1-specific motif. We and others have found that both domains are important for the interaction of Sgt1 with Hsp90 (23–26), which is required for assembly of the core kinetochore complex. This interaction is an initial step in kinetochore activation (24, 26, 27), which is conserved between yeast and humans (28, 29).We have recently shown that Sgt1 dimerization is important for Sgt1-Skp1 binding and therefore for kinetochore assembly (30). In this study, we have found that protein kinase CK2 phosphorylates Sgt1 at Ser³⁶¹, and this phosphorylation inhibits Sgt1 dimerization. Therefore, CK2 appears to regulate kinetochore assembly negatively in budding yeast.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]