Olga A. Petrova

Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase

Abstract The elongation of single-stranded DNA repeats at the 3′-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids—telomerase RNA, telomeric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the thermotolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the ‘fork’ at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.

NMR assignments of the N-terminal domain of Ogataea polymorpha telomerase reverse transcriptase

Telomerase is a ribonucleoprotein enzyme that adds telomeric DNA fragments to the ends of chromosomes. This enzyme is the focus of substantial attention, both because its structure and mechanism of action are still poorly studied, and because of its pivotal roles in aging and cellular proliferation. The use of telomerase as a potential target for the design of new anticancer drugs is also of great interest. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is essential for activity and processivity. Elucidation of the structure and dynamics of TEN in solution is important for understanding the molecular mechanism of telomerase activity and for the design of new telomerase inhibitors. To approach this problem, in this study we report the 1H, 13C, and 15N chemical shift assignments of TEN from Ogataea polymorpha. Analysis of the assigned chemical shifts allowed us to identify secondary structures and protein regions potentially involved in interaction with other participants of the telomerase catalytic cycle.

Identification of additional telomerase component of the yeast H. polymorpha is a step towards understanding the complex at the atomic level

59 Telomerase is a ribonucleoprotein complex whose function is to synthesize telomeres—repeated sequences at the ends of linear chromosomes. The absence of telomerase activity in the cell causes grad ual shortening of telomeres, with the onset of senes cence after reaching the critical length. In addition to the catalytic subunit and telomerase RNA, additional components are required for the functioning of this enzyme in vivo in the yeast S. cerevisiae cells, namely, Est1 and Est3. To date, structural data for these com ponents are absent due to the instability of the pro teins.

Chemical shift assignments and the secondary structure of the Est3 telomerase subunit in the yeast Hansenula polymorpha

Telomerase is a multisubunit ribonucleoprotein enzyme that is essential for continuous cellular proliferation. A key role of telomerase in cancer and ageing makes it a promising target for the development of cancer therapies and treatments of other age-associated diseases, since telomerase allows unlimited proliferation potential of cells in the majority of cancer types. However, the structure and molecular mechanism of telomerase action are still poorly understood. In budding yeast, telomerase consists of the catalytic subunit, the telomerase reverse transcriptase or Est2 protein, telomerase RNA (TLC1) and two regulatory subunits, Est1 and Est3. Each of the four subunits is essential for in vivo telomerase function. Est3 interacts directly with Est1 and Est2, and stimulates Est2 catalytic activity. However, the exact role of the Est3 protein in telomerase function is still unknown. Determination of the structure, dynamic and functional properties of Est3 can bring new insights into the molecular mechanism of telomerase activity. Here we report nearly complete 1H, 13C and 15N resonance assignments of Est3 from the yeast Hansenula polymorpha. Analysis of the assigned chemical shifts allowed us to identify the protein’s secondary structure and backbone dynamic properties. Structure-based sequence alignment revealed similarities in the structural organization of yeast Est3 and mammalian TPP1 proteins.

The genome-wide transcription response to telomerase deficiency in the thermotolerant yeast Hansenula polymorpha DL-1

BackgroundIn the course of replication of eukaryotic chromosomes, the telomere length is maintained due to activity of telomerase, the ribonucleoprotein reverse transcriptase. Abolishing telomerase function causes progressive shortening of telomeres and, ultimately, cell cycle arrest and replicative senescence. To better understand the cellular response to telomerase deficiency, we performed a transcriptomic study for the thermotolerant methylotrophic yeast Hansenula polymorpha DL-1 lacking telomerase activity.ResultsMutant strain of H. polymorpha carrying a disrupted telomerase RNA gene was produced, grown to senescence and analyzed by RNA-seq along with wild type strain. Telomere shortening induced a transcriptional response involving genes relevant to telomere structure and maintenance, DNA damage response, information processing, and some metabolic pathways. Genes involved in DNA replication and repair, response to environmental stresses and intracellular traffic were up-regulated in senescent H. polymorpha cells, while strong down-regulation was observed for genes involved in transcription and translation, as well as core histones.ConclusionsComparison of the telomerase deletion transcription responses by Saccharomyces cerevisiae and H. polymorpha demonstrates that senescence makes different impact on the main metabolic pathways of these yeast species but induces similar changes in processes related to nucleic acids metabolism and protein synthesis. Up-regulation of a subunit of the TORC1 complex is clearly relevant for both types of yeast.

Designing a system to test for the presence of any nontelomeric nucleotide at the 3′-chromosomal end

The number of telomeric repeats in telomeres correlates with the proliferative potential of eukaryotic cells. The lengthening of telomeres and telomerase activity are regulated in several ways. One of these mechanisms is based on the elongation of the 3′-chromasomal end of an additional nucleotide that is not a part of the telomeric repeat motif. This study presents an approach to testing for the presence of an additional nontelomeric nucleotide at the 3′-end of the G strand of chromosomes. The applicability of this method is demonstrated for thermotolerant yeast H. polymorpha. The possibility of modifying the method to test for the presence of an additional nucleotide at the 3′-end of the chromosomes of other organisms is discussed.

Towards structural characterization of H. polymorpha telomerase components

Telomeres are regions of non-coding DNA that cap the chromosomes, preventing the loss of coding DNA during cell division and contributing to chromosomal stability. In actively dividing cells, such as embryonic stem cells, the telomeres need to elongated by telomerase. The telomerase complex consist of the enzyme telomerase reverse transcriptase (TERT), telomerase RNA (TR) and additional proteins. TERT and TR are required for the telomerase activity in vitro. Telomerase is active in vast majority of the cancer cells ensuring continuous cell division and tumor growth. Syndromes leading to premature aging are often associated with short telomeres. Finding ways to regulate the telomerase activity would help to advance therapies for these conditions. However, the structural information available of the telomerase complex is very limited. We have chosen thermophilic yeast Hansenula polymorpha as a model system due to the stability of its proteins. The N-terminal domain of the TERT is essential for telomerase activity and possibly is involved in binding of TR, telomeric DNA and additional protein components of the telomerase complex. We have crystallised the N-terminal domain of H. polymorpha TERT and, in lack of a homologious structure, produced a selenomethionine derivative of the protein. MAD data on N-terminal domain has been collected to resolution of 2.0 Å at the PETRA-III beamline P13 (EMBL/DESY) in Hamburg. We will discuss the structure-function relationship of the N-domain and the whole TERT component.