Telomeric DNA replication

Telomeric DNA replication

  • DNA makes up all of the cells in our body. It is the genetic material that makes us who we are. Every organ in our body (skin, liver, heart, etc.) is made up of cells. The ends of linear chromosomes are called telomeres which protect genes from getting deleted as cells continue to divide. So telomeres are vital to our health.
  • Our cells replenish by copying themselves. This happens constantly throughout our lives. Telomeres get shorter each time a cell copies itself, but the important DNA stays intact.
  • Eventually, telomeres get too short to do their job, causing our cells to age and stop functioning properly.  Therefore, telomeres act as the aging clock in every cell.
  • When telomeres get too short, our cells can no longer reproduce, which causes our tissues to degenerate and eventually die.

The end problem of Linear DNA replication

Linear chromosomes have an end problem. After DNA replication, each newly synthesized DNA strand is shorter at its 5′ end than at the parental DNA strand’s 5′ end. This produces a 3′ overhang at one end (and one end only) of each daughter DNA strand, such that the two daughter DNAs have their 3′ overhangs at opposite ends.

Figure 1: The end problem of linear DNA replication

DNA polymerase cannot replicate and repair DNA molecules at the ends of linear chromosomes.

What is the reason behind this statement? (figure 2)

  • DNA polymerase need free 3’ OH group for adding nucleotides.
  • In linear chromosomes with multiple origins, the elongation of DNA in adjacent replicons provides a 3’ – OH group preceding each primer. So that once primer is removed DNA polymerase can fill the gap using DNA nucleotides. .
  • At the very end of a linear chromosome, however, there is no adjacent stretch of replicated DNA to provide this crucial 3’-OH group.
  • Once the primer at the end of the chromosome has been removed, it cannot be replaced with DNA nucleotides, which produces a gap at the end of the chromosome.
  • This single stranded region would be susceptible to enzymes that degrade single-stranded DNA.
  • The result would be that the length of the chromosome would be shortened after each round of replication and division. This is known as the end replication problem.

How the end replication problem solved during linear chromosome replication?

  • Replication of lagging strand only has this problem.
  • Eukaryotes have solved the end-replication problem by locating highly repeated DNA sequence at the end of each linear chromosome called telomere.
  • The action of the telomerase enzymes ensure that the ends of the lagging strands are replicated correctly.

End replication problem is not observed in circular chromosome (figure 2)

  • Most prokaryotes with circular genome do not have telomeres. During DNA replication, the leading strand of circular chromosomes can simply continue to grow from 5’→3’ direction until its 3’ end is joined to the 5’ end of the lagging strand coming around from other direction.
  • On the lagging strand, the RNA primer for the last Okazaki fragment can be replaced by the free 3’OH end of the leading strand coming around in the opposite direction.
  • In prokaryotes, the end-replication problem is solved by having circular DNA molecules as chromosome.
Figure 2

Lecture notes on telomere structure refer the following link

Link: http://easylifescienceworld.com/telomere/

Telomerase enzyme

  • It an enzyme in eukaryotic cells that adds a specific sequence of DNA to the telomeres of chromosomes after they divide, giving the chromosomes stability over time.
  • Telomerase was discovered in 1984 by Elizabeth Blackburn and Carol Greider of the University of California, Berkeley.
  • As DNA polymerase alone cannot replicate the ends of chromosomes, telomerase aids in their replication and prevents chromosome degradation.
  • Telomerase enzyme is a ribonucleoprotein (an enzyme with both protein and an RNA component)
  • Telomerase is a specialized reverse transcriptase that carries its own RNA template (provides the active site for RNA-dependent DNA synthesis).
  • The RNA part of the enzyme contains from 15 to 22 nucleotides that are complementary to the sequence on the G-rich strand.
  • This sequence pairs with the overhanging 3’ end of the DNA and provides a template for the synthesis of additional DNA copies of the repeats.
  • DNA nucleotides are added to the 3’ end of the strand one at a time and, after several nucleotides have been added, the RNA template moves down the DNA and more nucleotides are added to the 3’ end.
Figure 3: structure of telomerase enzyme

Mechanism of telomeric DNA replication (Involves 2 major events)

Here I am explaining the mechanism for the synthesis of telomeric DNA by Tetrahymena telomerase. The mechanism is similar for other eukaryotes too.

Event 1) Lengthening of the G rich strand

It occurs via 4 major steps:

Step 1: Binding of Telomerase to 3’ G rich tail

  • Telomerase recognizes the G-rich telomere sequence on the 3’ overhang of parent DNA strand.
  • Telomerase contains an RNA component that is complementary to the end of the G-rich strand and pairs with it. (Hybridization occurs between the TTG in 3′-end of the G-rich telomere strand and the AAC in template RNA of the telomerase in case of Tetrahymena).
  • Then the RNA component serves as a template for the addition of nucleotides onto the 3’ terminus of the parent DNA strand

(The short RNA component is 159 bases long in Tetrahymena. It includes a sequence of 15-22 bases that is identical to two repeats of the C-rich repeating sequence).

Step 2: Synthesis of telomeric DNA on G rich tail of parent DNA strand

  • Nucleotides are added to the 3’ end of the G-rich strand.
  • The telomerase uses the 3’—OH of the G rich telomeric strand as a primer for synthesis of tandem TTGGGG repeats.
  • After several nucleotides have been added, the RNA template moves along the DNA.
  • Telomerase adds G and T bases one at a time to the primer DNA as directed by the RNA template.
  • Telomere synthesis proceeds in 5’ to 3’ direction as usual.

Step 3:  Translocation of telomerase and rehybridization of template RNA with DNA

  • Having synthesized one copy of the repeat, the enzyme repositions to resume extension of the telomere.
  • The telomerase translocates to the new 3′-end of the telomere, pairing the left-hand AAC sequence of its template RNA with the newly incorporated TTG in the telomere.
  • The telomerase uses the template RNA to add six more nucleotides (GGGTTG, boldface) to the 3′-end of the telomere.

Translocation and Polymerization can repeat indefinitely to lengthen the G-rich strand of the telomere.

Step 4: Release of the telomerase enzyme from DNA

  • When the G-rich strand is made sufficiently long by adding several repeating units of the telomere, telomerase enzyme is released from the parent DNA strand.

(Telomerase does not fill the gap generated in the daughter DNA strand as a result of telomere replication.  It simply extends the 3’ end of the parent template strand).

Event 2) Filling of the C rich strand (4 sub steps under this event)

Step 1: Primer synthesis by primase enzyme

Primase can make an RNA primer complementary to the 3′-end of the telomere’s G-rich strand.

Step 2: DNA replication (DNA polymerase fill the gap)

DNA polymerase uses the newly made primer to prime synthesis of DNA to fill in the remaining gap on the C-rich telomere strand.

Step 3: Primer removal

  • When the primer is removed, it leaves a 12–16-nt overhang on the G-rich strand.
  • The removal of this primer once again leaves a gap at the 5’ end of the chromosome, but this gap does not matter, because the end of the chromosome is extended at each replication by telomerase; no genetic information is lost, and the chromosome does not become shorter overall.
  • The extended single-strand end may fold back on itself, forming a terminal loop by nonconventional pairing of bases

Step 4: Gap sealing by DNA ligase

Without telomerase activity, linear chromosomes would become progressively shorter. If the resulting terminal deletions extended into an essential gene or genes, this chromosome shortening would be lethal.

Figure 4: A) Telomeric DNA replication in Tetrahymena (ciliate) B) Telomere replication in human

 

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