Question

7.Review the structure of DNA. What is meant by a double-stranded structure with complementary anti-parallel strands?

8. What is the difference between replicating the leading strand and the lagging strand?

9. What is the role of each of the following in DNA replication: origin of replication, helicase, single-strand binding proteins, primase, DNA polymerase III, DNA polymerase I, ligase, and topoisomerase, Okazaki fragments, and telomerase?

10. What problem does the mechanism of DNA replication pose for the ends of linear eukaryotic chromosomes and what mechanism is used to solve this problem. How does that relate to mortality of normal cells?

Answer 1

7. DNA (Deoxy ribonucleic acid) is a nucleic acid present inside the nucleus of eukaryotic cells. Its one of the four major groups of biological macromolecules, i.e Carbohydrate, protein, lipid and nucleic acids. It is usually a double stranded structure. DNA composed of nucleotides and the two strands are joined by hydrogen bonds. In DNA, each nucleotide is made up of a ribose sugar (5carbon compound) called deoxyribose (where oxygen is removed from 2' carbon of the pentose sugar), a phosphate group, and a nitrogenous base.The nitrogenous bases are four types: adenine (A), guanine (G) cytosine (C), and thymine (T). The nucleotides are joined by phosphodiester bond. In DNA, the amount of A is equal to the amount of T, and the amount of C is equal to the amount of G. Usually turns in DNA is Right handed.

The double helixof the DNA is likened to a twisted ladder. The curving sides of the ladder represent the sugar –phosphate backbones of the two DNA strands; the joining are the base pairs. The spacing between base pairs is 3.32 Å, and the overall helix repeat distance is about 33.2 Å which means there are about 10 base pairs ( bp ) per turn of the helix.

The two strands are antiparallel. If one has 5' ?3' polarity from top to bottom, the other must have 3' ?5' polarity from top to bottom. The nucleotides in the two strands are complementary to each other.

8. Differences between leading strand and lagging strand

During DNA replication, a fork like structure is formed where one strand is synthesized continuously (leading strand) and another is synthesized through fragments (lagging strand).

Leading strand

In DNA replication, a leading strand is synthesized in the 5’-3’direction or the direction same as the replication fork movement. 3' end has free OH group which helps in addition of base pairs. It is synthesized continuously. This strand is formed as nucleotides are continuously added to the 3’ end of the strand after polymerase reads the original DNA template.

Lagging strand

A lagging strand is the strand which is synthesized in the 3’-5’ direction or opposite direction according to the movement of the replication fork. It is synthesized away from the replication fork. As it moves in the opposite direction, it is discontinuous and is synthesized in fragments. Interestingly, the primase, responsible for adding an RNA primer, has to wait for the fork to open before putting in the primer. The lagging strands have short discontinuous DNA fragments which are known as Okazaki fragments. These okazaki fragments are ligated afterwards by DNA ligase.

9.Role of the following :

• Origin of replication: Particular DNA sequences where initiation of replication will take place.
• Helicase: Helps to unwind the DNA and make it accessible for replication machinery to perform replication.
• Single-strand binding proteins: Also known as SSB proteins. It helps in stabilizing the unwinding DNA preventing the template from rewinding.
• Primase: It is an enzyme which adds a 5'-3' RNA primer at 3'end of the template DNA for DNA polymerase III to polymerize the strand.
• DNA polymerase III: DNA polymerase III adds neucleotides at a rate of around 1000 nucleotides per second. DNA Pol III activity begins after strand separation at the origin of replication.
• DNA polymerase I : Pol I mainly functions in the repair of damaged DNA
• ligase: promotes the joining of DNA strands together by catalyzing the formation of a phosphodiester bond
• topoisomerase: It helps in the overwinding or underwinding of DNAOkazaki fragments
• telomerase: It is responsible for maintenance of the length of telomeres of chromosome by adding guanine-rich repeat sequences. This enzymatic activity observed in gametes, stemcells and tumor cells.

10. In replication, a 5'-3' RNA primer is added to both the strands at their 3' end for DNA polymerase III to copy them from 5’ to 3’. At the end, this RNA primer is removed to be replaced with deoxy nucleotides. DNA pol I can replace the RNA primer only if its having a 3’ OH group preceding the primer. Thus, the end RNA primer (at telomeric position) lacks the preceding 3’ OH group and thus those sequences are missing in the newly synthesized DNA making the DNA shorter after every round of replication.

To overcome this problem, a riboenzyme name telomerase is being recruited. Telomerase has its own RNA (GT rich) sequence which binds complementarily with the DNA sequence at its 3’ end prior to where the RNA primer was bound. Thus, the 3’ end telomere is being elongated by 200-300 bases which will help to replicate the complimentary strand saving the 5’ end of parental strand.

Cells tend to lose 50- 100 telomeric sequences after each round of replication. This leads to exposure of the coding sequences to degrading enzymes and also causes loss of important genes, thus causing cell senescence and ultimately mediates cell death.