1.7.2. Telomere protection in somatic cell
Telomerase protection in somatic cell is done with the help of a group of protein complex known as Shelterin complex and protein in this group known as shelterin protein because those provide shelters to telomere. In mammals, these protein known as TRF1, TRF2, TIN2, POT1, TPP1, and RAP1. Shelterin act in specific way to remodel the telomere into a loop called a t-loop (for “telomere-loop”) and in t-loop each protein protects telomere in particular manner. TTAGGG repeat binding factor-1 (TRF1) binds to double-stranded telomere DNA with TTAGGG repeats. TRF2 paralog of TRF1 binds to the double-stranded parts of telomeres. Single-stranded telomeric DNA protected from endonucleases through binding of POT1 (protection of telomeres -1) to single-stranded 3’-tails of telomeres at just 2 nucleotides away from the 5’-end of the other strand in turn double-stranded telomeric DNA at 5’end of other strand also get protected from 5’-end exonucleases. TPP1 is a POT1-binding protein and present as partner of POT1 in a heterodimer. TIN2 (TRF1-interacting factor-2) plays an organizing role in shelterin complex, It connects TRF1 and TRF2 to TPP1/POT1 dimer. RAP1 (repressor activator protein-1) interact with TRF2. Shelterin proteins specifically present only at telomeres throughout the cell cycle and nowhere else.
1.7.3. Telomere structure and telomere-binding proteins in lower eukaryotes
Yeasts also have telomere-binding protein which protect the telomere ends by forming D-loop in which single strands not get hide and those protein bear a resemblance to mammalian shelterin proteins like Taz1 binds double-stranded telomere same as TRF. Taz1 with the help of Rap1 and Poz1 binds to a dimer of Tpz1 and Pot1 same asTPP1-POT1 dimer and protect single-stranded telomeric DNA. All these protein bond the telomere DNA up to 180 degrees by protein : protein interactions proteins bound to the double stranded telomere and also bound to its single-stranded tail.
Yeast Rap1 binds directly to double-stranded DNA show evolutionary relationship to mammalian Rap1. Rif1 and Rif2 are two partners of RAP1in yeast. Single-stranded telomeric tail binds by other protein complex Cdc13, Stn1, and Ten1. Ciliated protozoan Oxytricha in which telomere-binding proteins were firstly discovered having name TEBPa and TEBPb, which are evolutionarily related to POT1 and TPP1 in mammals binds with single-stranded 39-end of the organism’s telomeres and protect them from degradation. In schizosaccharomyces probe, Pot1 responsible for maintaining their integrity instead of limiting the growth of telomeres as mammalian POT1 does. Indeed loss of Pot1 can cause the loss of telomeres from this organism.
1.7.4. Telomeric protection through G-quadruplexes
Telomeres are protein–DNA structures at the ends of eukaryotic chromosomes that protect chromosome ends from fusion and are vital in safeguarding genomic stability. In Tetrahymena tandem repeats of short G-rich sequences (TTGGGG) present at 3′ strand of telomeres that project as a single-stranded DNA overhang and forming G-quarters, which is form in a cyclic array through synchronization or coordination of four guanine residue and multiple layers of this G-quarters leads to the formation of G-quadruplexes. This arrangement takes place through non Watson-Crick pairing or Hoogsteen hydrogen-bonding with the help of centrally positioned cation.
1.8. The Hayflick Limit
Normal human cell can be grown in culture upto 50 generations (or cycles of subculturing) for a limited period, after they enter a period of senescence, in which they slow down and afterward stop dividing, and lastly they attain a crisis stage and get die. This upper limit on the life span of normal cells is known as the Hayflick limit, discovered by Leonard Hayflick in 1960s. But tumor cells do not follow any such limit. Tumor cell divides generation after generation, for an indefinite period because. Tumor cell contain telomerase enzyme which replicates the end of DNA.
1.9. Modes of Replication
There are various mode of DNA replication - (1) Theta Replication, (2) Rolling Circle, (3) Linear Replication. Differ to each other with respect to the initiation and progression of replication, but all produce new DNA molecules by semiconservative replication.
1.9.1. Rolling-circle replication
It involves a single stranded intermediate that seems to roll off the plasmid. Plasmid’s double stranded DNA has positive and negative strand. In rolling circle replication double stranded ori that place within stem-loop structure get nicked by Rep A protein at positive strand and free 3’ OH serve as a primer which get replicate by DNA pol III in leading strand manner and displacing the 5’ end which is non-template strand by strand displacement reaction. The ends of the displaced strand are ligated to make a single stranded circular DNA molecule. RNA polymerase makes a primer at the 5’ end containing strand, thus also capable to act as template for lagging strand synthesis. After synthesis of new strand primer get remove by DNA pol I. The ends of the displaced strand are ligated by ligase to make a second double- stranded plasmid. E.g. M13, Φ 174, F plasmid.
Most plasmids use one of two common mechanisms of replication, both of which depend on the host cell replicative machinery for replication (DNA polymerases, ligases, primases, helicases, etc. are usually supplied by the host cell).
1.9.2. Theta replication –
It is normal mode of DNA replication which resembles the greek letter theta (θ) during replication. The theta mode of DNA replication is bidirectional in nature.
1.9.3. Linear replication –
This the type of replication takes place in linear chromosome like in eukaryotes.
1.9.4. Replication of organelle DNA (Models for mtDNA replication)
DNA polymerase γ is used exclusively for mtDNA replication and proofreading. Two models have been proposed for the mode of mtDNA replication, 1) strand displacement model, appeal to continuous DNA replication and 2) coupled model, proposes semidiscontinous DNA replication.
Strand displacement model
The strand displacement model (also called the strand asynchronous model) for mammalian mtDNA replication is the most widely accepted, longest standing model. In strand displacement model, replication is unidirectional around the circle and there is one replication fork for each strand in which One strand is called the heavy strand (H) and other strand is called light strand (L). The designation of the strands as H and L due to their different buoyant densities in denaturing CsCl density gradients centrifugation. Two origins (OH and OL) because here one priming event per template strand and it start within a region called the displacement or “D” loop of 500–600 nucleotide. The RNA primer synthesis by mitochondrial RNA polymerase. Firstly replication start on H strand starting at OH. When DNA polymerase polymerise approximately two-thirds of the mtDNA as a result replication fork get passes the major origin of L strand synthesis, thus exposing the site in single-stranded form at which new H strand synthesis start from OL. Synthesis is continuous around the circle on both strands. Multifunction endoribonuclease, RNase MRP cleave RNA primer. Same mechanism for cpDNA (cytoplasmic DNA) replication.
Strand coupled model
In strand coupled model at multiple sites lagging strand replication (L strand) get initiated, showing discontinuous synthesis of short Okazaki fragments thus requiring multiple primers. So, in this model the coupled lagging strand and leading (H strand) synthesis represents a semidiscontinous, bidirectional mode of DNA replication. The endoribonuclease, RNase MRP (mitochondrial RNA processing) has at least two functions, 1) removal of RNA primers in mtDNA replication, 2) pre-ribosomal RNA’s nucleolar processing. Mutation in RNase MRP cause cartilage-hair hypoplasia a rare form of dwarfism and due to its consequences multiple phenotypic manifestations (pleiotropy) occur which included short limbs, short stature, fine sparse hair, impaired cellular immunity, anemia, and predisposition to several cancers.