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Oxytricha nova telomere end-binding protein

Jay Galbraith '09 and Mark Luskus '10

Contents:

I. Introduction

The end-replication problem is a biological dilemma that all eukaryotes with linear chromosomes encounter.  Due to the inability of DNA polymerase to replicate DNA in the 5' to 3' direction, short single stranded regions of DNA on the ends of chromosomes are unable to be replicated, effectively shortening the chromosome over time. This continued loss of base pairs on the ends of chromosomes jeopardizes genetic information, threatening the integrity of the genome (Ohki, et al. 2001).  

Excessive chromosome shortening induces senescence, a state of irreversibly arrested growth.  Although the exact role of senescence is not understood, it is believed to play a role in both tumor suppression and cellular aging (Dimri, et al.  1995). Eukaryotic organisms with linear chromosomes have evolved telomeres as a mechanism to prevent the onset of senescence.  

Telomeres are sequences of repeated nucleotides - usually thymine and guanine - located at the end of linear chromosomes.  In addition to limiting the effects of the end-replication problem, telomeres also maintain the fidelity of the chromosome by protecting it against degradation from reactive oxygen species.  Telomeres also prevent two chromosomes from joining at the ends.  Additionally, the 3' overhangs of telomeres serve as binding sites for telomerase, an enzyme which lengthens telomeres and thus prevents early cellular senescence (Horvath, et al. 1998).

In the protozoan Oxytricha nova, as in many species, telomeres are bound to specialized proteins called telomere end binding proteins. The exact function of these proteins is uncertain; however, it has been hypothesized that they are important in higher-order telomere-telomere association, as well as protecting the integrity of the 3' telomere overhang (Peersen, et al. 2002).

II. General Structure


Oxytricha nova telemore end-binding protein (OnTEBP) is made up of two subunits; an alpha-subunit (56 kDa) and a beta-subunit (41 kDa).   The alpha-subunit itself is made up of two subunits: the N-terminal domain and the C-terminal domain .   The C-terminal domain interacts with a portion of the beta-subunit . The N-terminal domain interacts with ssDNA along with the beta-subunit .   The single stranded DNA takes on a non-helical structure .

III. Protein Folding


The structure of OnTEBP reveals the presence of four oligonucleotide/oligosaccharide binding (OB) folds . OB folds consist of a minimum of 5 β sheets that interact to form two orthogonally packed antiparallel β sheets, creating a β barrel like fold. Two OB folds can be found in the α N-terminal domain, one that encompasses residues 225 to 299 and another that encompasses residues 52 to 149 . These OB folds closely associate to form a complex ligand-binding surface via variable loops connecting β strands in two sets of crossing β sheets. This pairing is essential to form specific interactions with ssDNA, as single protein folds are insufficient to create specific interactions with ssDNA. Additionally, these OB folds exhibit similar ssDNA nucleotide binding due to the cooperative relationship between their ssDNA recognition loops .

The C-terminal domain of the α subunit is composed of an OB fold that is complexed with the oligopeptide loop of the βsubunit . The C-terminal domain of the α subunit, due to the OB fold and β loop complex, is a unique structure that suggests that the full complexity of OB folds is not yet known .
 
The final OB fold is the largest component of the β subunit. Encompassing residues 41 to 119 , it can be distinguished by its loosely folded core , which differs from the barrel structure of the other OB folds, as well as the unique orientation of the α helix, which is 90 degrees relative to the position of helices in other OB folds. Since the β subunit makes critical contacts with ssDNA and the α subunit that maintain the structural integrity of the complex, it is theorized that these unique structural properties facilitate the incorporation of β into the complex is dependent on the presence of both ssDNA and the α subunit.

IV.Protein-Protein Interface



The interface between the α and β subunits involves 53 residues of α and 49 residues of β. The points of contact between the α and β subunits are in helix Cß , which spans the N- and C- terminal domains of α, and the ß subunit peptide loop , which interacts along the surface of the C-terminal domain of α . There are a number of polar charged amino acid residues on the surface of helix Cβ that establish a network of hydrogen bonds with polar residues on the α subunit. Two leucine side chains, L236α and L330α interact hydrophobically with the aliphatic portions of the polar charged residues on helix Cβ. Helix Cβ also contains residues L156β, I160β, and V164β , which pack into a hydrophobic groove in the α C-terminal domain. 

The ß subunit peptide loop is bound to the α C-terminal domain via similar leucine, isoleucine, and valine residues.  These interact with a number of hydrophobic residues on the α C-terminal domain , including Tyr, Leu, Phe, Pro, Gly, and Ala, effectively anchoring the α and β subunits together. 

V. Protein-ssDNA interactions


The OnTEBP interacts with three regions of telomeric ssDNA . Beginning on the 3' end, a hydrogen bond forms between the 3' hydroxyl group and the peptide amide of Lys66 on the alpha-domain . Guanine 12 shows ionic interactions with Lys261, as well as base stacking with the deoxyribose sugar of G11 and the base of T8 . G11, in addition to base stacking with G12, is held between the OB folds in the alpha N-terminal domain, and forms a hydrogen bond with the peptide carbonyl group of Lys261 . Guanine 10 has several interactions with both domains of OnTEBP, base-stacking between Tyr239 in the alpha-domain and Arg140 in the beta-domain, hydrogen bonding to Lys145 side chain in the beta domain, and interacting with a Leu258 side chain in the alpha-domain . This shows how important G10 is to the stability of the protein complex. Nucleotide G9 interacts only with the beta-domain, packing against a group of hydrophobic amino acid side chains, and forming a hydrogen bond with Glu45 . Thymine 8 stacks with G12 and makes a hydrogen bond with Tyr72 on the alpha-domain . 

The next region of ssDNA shows a great deal of stacking, both within the bases and with amino acid side chains . Thymine 7 stacks with both G9 and T6 . T6 stacks with His292 on the alpha-subunit . This stacks with Tyr293, which stacks with G4 .
 
The third region of the telomeric ssDNA is located at the 5' end . Arginine 274 in the alpha subunit plays a critical role in this region, interacting with the ssDNA in multiple ways: it forms a hydrogen bond with G4, a hydrogen bond with the phosphodiester backbone of G3, and base stacks with G3 . Additionally, G3 and G4 interact with Asp223, Asp225, and Lys77 on the alpha-subunit through hydrogen bonding . The G2 base fits between Tyr130 and Tyr103 of the alpha-domain, and hydrogen bonds with Gln135, also on the alpha-domain . The contacts between G1 and the protein were not visible due to the way the protein complex crystallized.

VI. References

Horvath, MP, VL Schweiker, JM Bevilacqua, JA Ruggles, SC Schultz.  1998.  Crystal Structure of the Oxytricha nova Telomere End Binding Protein Complexed with Single Strand DNA.  Cell 95: 963-974.

Peersen, OB, JA Ruggles, SC Schultz.  2002.  Dimeric structure of the Oxytricha nova telomere end-binding protein α-subunit bound to ssDNA.  Nature 9(3): 182-187.

Reiko Ohki, Toshiki Tsurimoto, Fuyuki Ishikawa.  2001.  In Vivo Reconstitution of the End Replication Problem.  Molecular and Cellular Biology 21(17): 5753-5766.

GP Dimri, X Lee, M Acosta, G Scott, C Roskelley, EE Medrano, M Linskens, I Rubelj, O Pereira-Smith.  1995.  A biomarker that identifies senescent human cells in culture and in aging skin in vivo.  PNAS 92(20): 9363-9367.  

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