The Human Telomere
Telomeric DNA (5’-TTAGGGn -3’) is found at the end of the chromosome and serves to protect the coding regions of chromosomes from degradation due to the end replication problem. In adult somatic cells, single stranded telomeric overhands are cleaved by DNA repair enzymes after every replication cycle, thus shortening the chromosome. Once the telomeres reach a critical length after so many replications, apoptosis is triggered killing the cell before coding regions of the chromosome can be damaged. On average, these chromosomal ends lose 50 bases per replication cycle. During infancy and childhood, these ends can be re-extended by the reverse transcriptase enzyme, telomerase; however, in adult cells, telomerase is down-regulated. In the majority of known cancers, telomerase has been shown to be upregulated, subsequently allowing cancer cells to continuously replicate as the telomeric regions of chromosomes are re-extended making them ‘immortal’. In order to overcome this pathway, treatments utilizing chemotherapeutic agents can still induce cancer cells to undergo apoptosis. Apoptosis results in the release of two major apoptotic nucleases: capase-activated DNase (CAD) and endonuclease G (endo G) that can fragment and destroy the majority of DNA from the apoptotic cell. However, the telomeric sequence is able to fold into the nuclease resistant quadruplex secondary structure and we have found that these fragments are able survive apoptotic degradation and are released into the cell free matrix. Because telomeres play such a critical role in cell aging, cancer, and other age related diseases; we believe that these cell free telomeric fragments released from apoptotic cells may also play a biological role in immunity pathways, such as those governed by Toll-Like Receptors.
The Quadruplex Structure
First predicted by D. R. Davies in 1962, this non-helical structure exists when repetitive runs of guanines (minimum of three) are separated by short segments of single stranded nucleotides. As shown in Figure 1a, this structure occurs when four guanines, upon close proximity, form Hoogstein base pairs forming a square with a coordinated counter cation in the center (either potassium or sodium). These planar tetrads can then stack upon other tetrads to form a structure as shown in Figure 1b. These G-quadruplex structures are more thermodynamically stable than their duplex counterparts and are often found to be nuclease resistant. Often a G-quadruplex forming sequence will be found in oncogene promoter regions and are believed to play significant roles in the expression of cancer causing genes such as the c-MYC and B-cell CLLymphoma 2 (Bcl-2) genes.
Depending on the number of strands, salt conditions, and loop sequence these quadruplexes can fold into a variety of topological structures as depicted in Figure 2.
For many years there has been debate in the scientific community as to whether or not the quadruplex structure actually exists in vivo or is just an artifact of in vitro studies. However, the existence of these structures in the cell has received growing support from recent studies utilizing fluorescent probes or antibodies that can bind specifically to quadruplex structures within the cell. In light of this recent evidence and the fact the a quadruplex folding sequence can be found in a number of oncogene promoter regions as well as in telomeres, there has been a push in the scientific community to further research targeting the quadruplex structure for anti-cancer drug development as well as increase understanding of the biological role this structure must play.
Figure 3. Immunofluorescence of a quadruplex structure specific probe, BG4, on metaphase chromosomes of HeLa cells. The red foci of BG4 were observed in interstitial regions (i, ii, iii) and at the telomeres (iv, v). The DNA was counterstained with DAPI (blue) and the scale bar corresponds to 2.5 μm. This figure was adapted from Biffi, G. et al. Nat Chem. Mar 2013; 5(3): 182–186.
As can be seen from above, understanding the structural and thermodynamic properties of the quadruplex structure will further our understanding of the human telomere as well as how telomeric fragments within the cell free matrix may behave. In my previous research as a graduate student I found that different length human telomere fragments have different thermodynamic and structural properties and this may play a vital role in the biological function of these cell-free telomeric fragments. It is my plan as a research mentor to continue basic structural and thermodynamic characterizations of different length human telomere fragments ranging from 12 to 200 bases. Currently, the 22-24 base length telomere models have been well characterized using NMR, X-ray crystallography, DSC, CD and UV-Vis studies, however, this length only encompasses one quadruplex unit. I believe that these cell-free fragments released from cells may also exist in longer or shorter fragments, and little is understood about how these lengths behave in vitro. To study this, I will employ structural characterization techniques such as NMR, CD and X-ray crystallography and thermodynamic characterization techniques such as DSC, ITC, UV melts, CD melts, and PPC-DSC, all of which can be done at UAB with the given support from my mentor and chair of the chemistry department, David Graves. This research project will give my students valuable hands on experience with a wide range of instrumentation and can be easily modified to meet the work demands of each individual student who is interested in participating.