Question

What does “semi-discontinuous” DNA replication mean? Explain in words how the lagging DNA strand is replicated in bacteria. What specific enzymatic activities does DNA polymerase I have? Name each activity and explain its role in DNA replication. If DNA polymerase I lacked its 3’-5’ exonuclease activity, what specific consequences with respect to DNA replication would be observed? What consequences would be observed if, instead of DNA pol I, DNA polymerase III lacked its 3’-5’ exonuclease activity? Explain your answers.

Answer 1

(i) "Semi-discontinuous" DNA replication means that synthesis of one daughter strand takes place continuously as replication is continuous in this strand whereas the synthesis of other daughter strand takes place in discontinuous manner. The replication is continuous on one template strand with polarity 3'5' which is known as leading daughter strand. the replication is discontinuous in the form of short okazaki fragments on other template strand with polarity 5'3', which is known as lagging daughter strand.

(ii) The initiation of DNA elongation starts when the primer strand is synthesized opposite to the parental strand. The primer is 5-10 nucleotide long, serves as a stepping stone to start errorless replication which is synthesized by RNA primase. Once the primer strand is formed, DNA replication occurs in 5'3' direction, i.e during synthesis of a new strand, deoxyribonucleoside triphosphates are added only to the free 3'OH end. Thus the nucleotide at 3' end is always the most recently added nucleotide to the chain. During DNA replication, the DNA dependent DNA polymerases catalyze polymerisation only in one direction, that is 5'3'. This creates some additional complications at the replicating fork. Consequently, the replication is continuous towards the replication fork on one template strand with polarity  3'5' as polymerase can add nucleotides in this direction. This continuously synthesizing strand is called leading strand. The other strand which is complementary to 5'3' parental strand, synthesize away from the replication fork in a discontinuous fashion such that the polymerase has to move back toward the replication fork to add bases in the new primer in the direction away from the replication fork. This step produce short pieces of DNA known as Okazaki fragments and the strand having it is called lagging strand which is synthesized in a discontinuous manner.

(iii) DNA polymerase is also known as kornberg enzyme and an important class of polymerase enzyme required in DNA replication. It has got both exonuclease and polymerase activity. It can be stated as follows: (a) Exonuclease activity- It has exonuclease activity in both 5'3' and  3'5' direction. The  3'5' exonuclease activity meadiates proofreading and 5'3' exonuclease activity mediates nick translation during DNA repair. It removes RNA primer through 5'3' exonuclease activity and replace it with the nucleotide of DNA and can correct the thymidine dimer (T=T) formed under the influence of UV-rays. It also takes part in repair replication. (b) Polymerase activity- It polymerizes in 5'3' direction and can adds 1000 nucleotides per minute in a DNA dependent DNA polymerase activity. It has also got 5'3' RNA-dependent DNA polymerase activity.

(iv) DNA polymerase I use its exonuclease activity in 3'5' direction to mediate proofreading. When an incorrect paired nucleotide is added, DNA polymerase I recognize it and excises the mismatched base and correctify it by replacing the mismatched base and reinserting the correct base so that replication continues. If DNA polymerase I lacked 3'5' exonuclease activity, then the proofreading mechanism will be hampered resulting in DNA damage.

(v) In case of DNA polymerase III, if it lacked 3'5' exonuclease activity then also the proofreading mechanism will become non-functional. All the DNA polymerases have exonuclease activity in 3'5' direction. Proofreading is effective because DNA polymerase requires a primer and is not able to initiate DNA synthesis, i.e it is unable to deposit the first nucleotide in a daughter strand without the primer.