DNA replication is carried out by DNA polymerase. During the replication, one DNA strand synthesized as leading strand and the other as lagging strand. The leading strand synthesis is continuous, whereas lagging strand synthesis is discontinuous. DNA replication at the DNA ends i.e. telomeres is an altogether different story. If a primer forms at the end of a linear DNA molecule to initiate replication of the leading strand, a single-stranded gap remains at that end after DNA has been synthesized and the primer has been removed. DNA pol cannot fill his gap as no 3‘-OH group is available onto which nucleotides can be added. If this gap were left unfilled a linear DNA molecule would shorten at each end by the length of the primer with each replication cycle. After many rounds of replication, the molecule would disappear. This problem is known as the linear DNA replication paradox. Besides, as the lagging strand is replicated in the discontinuous manner, it cannot be replicated in its entirety. When the RNA primer is removed, there is no upstream strand onto which DNA pol can build to fill the resulting gap. Without some special mechanism, the daughter DNA strand resulting from lagging strand synthesis would be shortened from the telomeric ends at each cell division.
The enzyme that prevents this shortening of DNA strands is known as telomerase. The enzyme functions as a reverse transcriptase (RNA-dependent DNA polymerase). It holds a small RNA molecule as its component. It was discovered by Elizabeth H. Blackburn et al. The enzyme contains a catalytic site that polymerizes the nucleotides using RNA as the template. The RNA template is part of the enzyme and is approximately 160 bases long. The enzyme uses this RNA as template for adding telomeric repeats to the chromosome ends. First, the 3’ end of the telomere hybridizes with the RNA component of telomerase. Within the RNA sequence is the 6-nucleotide repeated sequence 5’CCCCAA3’ (Tetrahymena) which is complementary to the telomere repeat sequence. Telomerase binds to the 3’ end of to the repeated region. Telomerase binds to the 3’ end of the ssDNA, adding new copies of the TTGGGG repeat sequence. These repeats further extend the single stranded gap left after primer removal. Primers may be added to the repeated segments of DNA and a DNA pol may now fill in much of the gap with DNA.
The repetitive sequence added by telomerase is determined by the RNA associated with the enzyme, which differs among telomerases from different organisms. This extends the dsDNA till it is at least as long as, or longer, than the parent molecule. The single stranded end left after several rounds of addition can be trimmed. The extension and trimming of telomeric DNA is not precise and therefore the number of telomeric repeats is highly variable. Certain proteins, like Rap1 in S. cerevisiae, TRF1 in human beings, keep track of the number of telomeric repeats. It is actually the number of protein molecules attached to the telomere ends that is counted, and not the number of repeats, to decide if telomeres should be added.
The activity of telomerase gradually declines indicating that the DNA molecules must grow shorter with each cell division. This is of little consequence for most cells as they undergo a limited number of cell divisions and then stop dividing: This is the normal ageing process which occurs in all body cells, end at a certain telomeric length, and cells stop dividing. However, if telomerase starts behaving erratically, the chromosomes will not be shortened and cells will continue to divide leading to continuous growth. When cancer cells were examined for the presence of telomerase, the active enzyme was found in abundance, indicating that the linear DNA molecules were being fully replicated. Thus telomerase is found to be essential for cancer cells to continue dividing. Thus, the attention is now turning to the possible clinical application of this knowledge. Research is now under way to determine if blocking the activity of telomerase may be an effective treatment for cancer. Further, studying normal telomere shortening, which acts as a biological clock, may help understand senescence.