MIC115 Recombinant DNA Cloning Lecture 18 Telomere PDF

MIC115 Recombinant DNA Cloning 2024FQ Lecture 18 – Telomere Maintenance in Human Cells LECTURE 18 LEARNING GOALS 1. Define key features of human telomeres: what are the sequences; where are they on the chromosome;...

MIC115 Recombinant DNA Cloning 2024FQ Lecture 18 – Telomere Maintenance in Human Cells LECTURE 18 LEARNING GOALS 1. Define key features of human telomeres: what are the sequences; where are they on the chromosome; which enzyme extends them; what protein complexes bind them. 2. What are the core components of telomerase? What are the key features of telomerase- directed DNA synthesis (template, primer, synthesis direction, etc.)? Why is telomerase essential for the continued proliferation of human cells? 3. Describe the main difference of telomerase biology between human and mouse. 4. Describe different ways to disrupt telomerase function. 5. Understand the principle and describe the basic procedure of immunostaining and FISH. An overview of telomere maintenance in human cells In eukaryotic cells, telomeric DNAs locate at the very end of each linear chromosome. Telomeric DNA is made up of simple tandem repeats. Human telomeres contain hundreds to thousands copies of TTAGGG repeats within its double-stranded region followed by a short (100-200 bases) G-rich 3’-overhang. Telomeric DNAs are extended by telomerase, a ribonucleoprotein complex that is composed of a reverse transcriptase that provides the catalytic function, an RNA subunit that provides a short template region to direct the synthesis of telomeric DNA, and several accessory protein subunits that stabilize the telomerase complex. Telomerase is not expressed in most human somatic cells. But expressed in germ line cells, stem cells, and majority of human tumors. Telomeric DNAs are bound by telomeric sequence-specific binding proteins which are collectively called the shelterin complex (you don’t need to memorize the name of individual subunits of the shelterin complex). One major function of telomeres is to distinguish normal chromosome ends from double- stranded DNA breaks, thereby protecting chromosome ends from being processed by the cellular DNA repair machinery. Another major function of telomeres is to allow the complete replication of linear chromosomes, solving the end replication problem and preventing loss of the genetic materials located at or near chromosome ends during cell proliferation. The formation of the telomerase holoenzyme is a multistep process. It is thought that the assembly of telomerase is completed in the Cajal Bodies (CBs). CBs are membrane-less organelles and largely consist of proteins and RNA. In S phase of the cell cycle, telomerase is recruited to telomeres through an interaction between telomerase and a shelterin component. At G1 and G2 phases of the cell cycle, telomerase stays in the CBs. In normal human somatic cells, telomerase expression is absent or too low to maintain telomeres. When these cells divide, because of the end-replication problem and nucleolytic attack by cellular nucleases at chromosome ends, cells’ telomeres keep shorten. When telomeres reach a critical length, DNA damage signaling pathways are activated and cells enter replicative senescence (i.e., cellular senescence). Replicative senescence is a state in which the cells irreversibly exited the cell cycle, becoming large and flat but retaining metabolic viability. 1 MIC115 Recombinant DNA Cloning 2024FQ Cellular senescence is considered a major protection mechanism to prevent somatic cells from dividing uncontrollably and becoming cancerous. In human stem cells and cancer cells that have active telomerase, telomeric DNA gets extended by telomerase. During every cell cycle, telomerase elongates while nucleolytic attack and end- replication problem shorten telomeres. The elongation and shortening reaches an equilibrium and telomere length is maintained in a homeostasis state at a stable length. As a result, these cells can keep dividing. Telomerase inhibition, however, leads to cell death (apoptosis) or growth arrest in these cells. Therefore, inhibition of telomerase function has been explored as an anti-cancer therapeutic strategy. An important difference between human and mouse telomere and telomerase biology: telomerase is active in mouse somatic cells and telomerase activation is not a critical step for mouse tumorigenesis. Therefore, mouse model is not ideal for study of telomerase activity control during cancer development. Such studies are usually conducted using 2D or 3D cultures of human cells. Microscopy A. Light microscopy Light microscopes consist of a series of solid-state lens that can magnify up to 1000x: A combination of objective lenses, tube lenses and eyepiece lenses are arranged to focus the image of an illuminated specimen into the eye. B. Fluorescence microscopy a. Cellular components can also be stained with fluorescent dyes and examined by fluorescence microscopy. Fluorescent molecules usually absorb light at one wavelength and emit it at another, longer wavelength. b. (FYI) The optical light path for a fluorescent microscope is similar to an ordinary light microscope except that the illuminating light, from a very powerful light source, is passed through two sets of filters: one to filter the light before it reaches the specimen and one to filter the light obtained from the specimen. The first filter passes only the wavelength that excite the fluorescent dye, while the second filter blocks out this excitation light and passes only those wavelengths emitted when the dye fluoresces. c. Immunofluorescence staining to detect protein in cells/tissue 1) Subcellular locations of different protein molecules can be examined by immuno- fluorescence staining followed by fluorescence microscopy. Typical steps: i. a chemically fixed, permeabilized sample (cells or tissue) is incubated with a primary antibody that binds specifically to the protein of interest. ii. The sample is next incubated with a fluorescent dye-labeled secondary antibody that specifically binds to the primary antibody at multiple places. iii. The sample can be examined by fluorescence microscopy. 2) This detection is very sensitive because multiple molecules of secondary antibody recognize each molecule of primary antibody. 2 MIC115 Recombinant DNA Cloning 2024FQ 3) Joining of different dye-conjugated antibodies permits different proteins (molecules) to be visualized in the same specimen. For immunofluorescence staining, cells/tissues are usually fixed with a chemical crosslinker to ensure cell components remain in place; cells are also permeabilized with a detergent to permeabilize membranes to allow antibody access to cell components. d. Fluorescence in situ hybridization (FISH) to detect nucleic acids in cells/tissue Oligonucleotide probe labeled with a fluorescent dye can be used to visualize a gene or mRNA of specific sequence directly in a cell or in tissue via nucleic acids hybridization. 3