t-loop formation at single molecule level.

The average lifespan of a cell is approximately 50 cycles after which the cells go into senescence, inability to replicate. Early published work clearly suggests that the growing cells have an inherent knowledge of the number of cycles they have divided and this attribute of the cells is very much dependent on the structures on the end of the chromosomes known as the telomeres. These structures at the end of the chromosomes are known to shorten after every cell cycle. Once the telomere reserve has run out, cells stop dividing. Telomeres play important roles in maintaining the stability of linear chromosomes. The telomeric structure allows a cell to distinguish between natural chromosome ends and double-stranded DNA breaks. Telomere dysfunction and associated chromosomal abnormalities have been strongly associated with age-associated degenerative diseases and cancer. Telomere maintenance involves dynamic actions of multiple proteins on a long complex DNA structure. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure-function relationships that govern telomere maintenance. These telomeres form little loops at the end of the chromosomes, which are called the t-loops. These are formed by inserting the ends of the chromosome, which is usually 3’ overhang back into the DNA of the chromosome. Thus, very short telomeres, which is the scenario in old aging cells or sometimes cancer cells, can no longer form t-loops. The exposure of these chromosome ends, 3’ overhangs which cannot be inserted back into the DNA of the chromosome, would alert the cells and thus stop cells from dividing. If we can elucidate the mechanism of this t-loop formation, we can introduce methods to stop shortening of these telomeres and make drugs to stop these processes.
There were two talks this year focusing on the t-loop formation at the single molecule level. While Xi Long talk in the DNA structure session used Magnetic tweezers to understand this, Hong Wang’s talk in the Nanoscale Biophysics session on Saturday talked about these structures , using new technique developed in their lab , DREEM ( Dual Resonance Enhanced Electrostatic force Microscopy). Xi long et al was able to show the melting of telomeric DNA substrates on applying torque and the binding of these substrates with the single stranded oligos but the major drawback in her work being the absence of TRF2 protein from the shelterin complex, which has been shown to be required for the formation of t-loops. Hong et al was able to predict a possible mechanism of the t-loop formation based on the interaction of the telomeric DNA with the shelterin protein, very cool work and technique , actually showing the DNA inside the protein DNA complexes. How cool to be able to see what happens inside the protein DNA complex!!!
Would be looking forward to BPS 2016 for their work to better understand this mechanism…


DNA Methylation at single molecule level

Just heard this talk….Nanomechanics of DNA Methylation!!! Always drew my interest as it is one of the major epigenetic modifications leading to gene silencing. Gene silencing occurs when a cell develops or exercises the ability to prevent or reduce the expression of a certain gene. These modifications do not alter the sequence of the DNA but they affect how the genes are read by the cell. Every tumor without any exceptions, has varied frequencies of DNA methylation pattern, indicating an altered genome. No doubt, it has been studied to be a cause of breast cancer, ovarian cancer, colorectal cancer, head and neck cancer, aging, Rett Syndrome and many more. Already epigenetic therapies towards drug development involving cancer is driven by DNA methylation inhibitors (FDA approved) like 5-azacytidine (Vidaza), 5 aza-2/-deoxycytidine, Decitabine (Dacogen) and there are many more in various phases of clinical trials. Henceforth, seeing this at a single molecule level becomes even more exciting. There have been recent papers studying these, but the one which almost resembled the work done by Csaba I. Pongor was by Kaur et al “Hydrophobicity of methylated DNA as a possible mechanism for gene silencing.” Physical biology 9, no. 6 (2012): 065001. While both were able to observe a shorter contour length in the methylated form of the DNA, and an increase in persistence length in the methylated form, but the interpretations seemed quite different, Csaba’s work had in additional a lowering of stress modulus on methylation. I would have certainly liked to observe a titration study on the degree of methylation and observed the effects, likewise done by kaur et al, but still beautiful work!! Well it is indeed fascinatinig how just adding a single methyl group to the DNA can give such huge variations to it. Surely more work needs to be done at single molecule level to understand this mechanism further and I would be looking forward to it.