In this article we will discuss about the Metabolism of DNA:- 1. DNA Synthesis 2. Replication of DNA 3. Degradation 4. The DNA Polymerase Complex 5. Enzymes Repair Damaged DNA.
- DNA Synthesis
- Replication of DNA
- Degradation of DNA
- The DNA Polymerase Complex
- Enzymes Repair Damaged DNA
1. DNA Synthesis:
a. The initiation of DNA synthesis requires priming by a short length of RNA (10-200 nucleotides long).
b. This priming process involves the nucleophilic attack by the 3′-hydroxyl group of the RNA primer on the α-phosphate of the deoxynucleoside triphosphate with splitting off pyrophosphate.
c. The 3′-hydroxyl group of the recently attached deoxyribonucleoside monophosphate is then free to carry out a nucleophilic attack on the next entering deoxyribonucleoside triphosphate on its α-phosphate moiety with the splitting off pyrophosphate.
d. The selection of proper deoxyribonucleotide is dependent upon proper pairing with the other strand (template) of DNA molecule.
e. The template dictates in which dNTP is complementary and holds this by hydrogen bonding.
f. The polymerization of deoxyribonucleotides takes place by such a process in a discontinuous phase of about 100 nucleotides in length.
g. This newly synthesized DNA strand attached to RNA primer is called okazaki fragments.
h. When many okazaki fragments are generated, the replication complex begins to remove the RNA primers by DNA polymerase I and the gaps left by their removal are filled up by proper base paired deoxynucleotide. The enzyme DNA ligase seal the fragments of newly synthesized DNA.
2. Replication of DNA:
a. Due to unwinding of double helix of DNA, each strand acts as a template for the formation of a new strand. This process is called replication.
b. Types of Replication:
(i) Conservative replication:
The parental strand is never completely separated. So, after one round of replication, one daughter duplex contains only parental strands and the other only daughter strands.
(ii) Semiconservative replication:
The process of unwinding of the double helical daughter molecules, each of which is composed of a parental strand and a newly synthesized strand formed from the complementary strand, called semiconservative replication.
b. Process of Replication:
(i) Initiation of DNA replication:
(1) Replication begins at a specific initiation point and this is a unique sequence of bases called Ori.
(2) In Ori there are two series of short repeats such as three repeats of a 13- base pair sequence and four repeats of a 9-base pair sequence.
(3) In the initiation process about 20 Dna A protein molecule each with a bound ATP, bind at the four repeats of 9-base pair sequence, DNA is wrapped around the complex (initial).
(4) With the help of ATP and histone like protein HU, the three 13-base pair repeats are denatured to give open complex.
(5) With the help of Dna C protein ATP Dna B protein binds to the open complex to form prep riming complex. As a result, unwinding of DNA occurs and priming replication starts.
In the elongation process of replication two operations occur such as leading strand synthesis and lagging strand synthesis.
(1) Leading strand synthesis:
(a) Leading strand synthesis begins with the synthesis of RNA primer (10 to 60 nucleotides) by Dna G protein at the replication origin.
(b) Deoxyribonucleotides are then added to this primer by DNA polymerase III (it keeps pace with the replication fork).
(c) SSB molecules help to stabilize the separated strand.
(d) Helicases separate the two DNA strands at the fork.
(e) Topoisomerase II (DNA gyrase) acts to relieve the stress generated by helicases.
(f) This leading strand is replicated in a continuous manner in the 5′ to 3′ direction.
(2) Lagging strand synthesis:
(a) Lagging strand is replicated discontinuously and must be accomplished in short fragments (okazaki fragments) synthesized in the direction opposite to fork movement.
(b) Synthesis of okazaki fragments:
The multi-protein primo-some complex travels in the same direction as the replication fork.
At intervals, Dna G protein synthesizes an RNA primer for a new okazaki fragment. The synthesis proceeds in the direction opposite to fork movement. Each primer is extended by DNA polymerase III.
(c) When the new okazaki fragment is complete, the RNA primer is removed by DNA polymerase I. The remaining nick (a phosphodiester bond broken to leave a free 3′ OH and 5′ phosphate) is sealed by DNA ligase.
Very little is known about this process, it is assumed that DNA topoisomerase IV appears to be necessary for final separation of the two completed circular DNA molecules.
3. Degradation of DNA:
DNA damage may be classified into 4 forms:
a. Single Base Alteration.
b. Two Base Alteration.
c. Chain Breaks.
(a) Single Base Alteration:
The one base damage includes the hydration of the cytosine residue by ultraviolet irradiation.
(b) Two Base Alteration:
The two base damage includes Thymine-thymine dimer formation via a cyclobutane moiety.
(c) Chain Breaks:
Chain breaks may be created by irradiation such as X-ray exposure.
Cross-linkage agents which link bases of opposite strands also induce 2 base alterations. Crosslinks can also occur between the DNA molecule and histones.
4. The DNA Polymerase Complex:
a. The different DNA polymerase molecules engage in DNA replication.
These are involved in three important properties:
(i) Chain Elongation
(ii) Processivity, and
b. Chain elongation shows the rate at which polymerization occurs. Processivity is an expression of the number of nucleotides added to the nascent chain before the polymerase disengages from the template. The proof-reading function identifies copying errors and corrects them.
c. InE. Coli, polymerase 111 (Pol 111) functions at the replication fork. It catalyzes the highest rate of chain elongation and is the most processive. It is capable of polymerizing 0.5 Mb of DNA during one cycle on the leading strand. It is the product of Dna E gene in E. Coli.
d. Polymerase 11 (Pol 11) is mostly concerned with proof-reading and DNA repair.
e. Polymerase 1 (Pol 1) completes chain synthesis between okazaki fragments on the lagging strand.
f. In mammalian cells, the polymerase is capable of polymerizing about 100 nucleotides per second. This reduced rate is the result of interference by nucleosomes.
5. Enzymes Repair Damaged DNA:
a. It has been concluded that surviving species have the mechanisms for repairing DNA damage caused as a result of replication errors or environmental insults.
b. Replication depends on the specific pairing of nucleotide bases. Proper pairing is dependent upon the presence of favoured tautomer’s of the purine and pyrimidine nucleotides. The proper base pairing can be assured by monitoring the base pairing twice.
Such double monitoring occurs in both bacterial and mammalian systems. This double monitoring does not produce errors of mis-pairing due to the presence of the un-favoured tautomer’s.
c. Replication errors lead to the accumulation of mutations.
d. DNA damages are replaced by mismatch repair, base excision repair, nucleotide excision repair, and double strand break repair. The defective in one strand can be returned to its original form by relying on the complementary information stored in the unaffected strand.
Some Repair Enzymes are Multifunctional:
i. Some repair enzymes are found as components of the large TF11H complex that plays central role in gene transcription. Another component of TF11H is involved in cell cycle regulation. Some repair enzymes are involved in gene rearrangement that normally occur.
ii. In patients with ataxia-telangiectasia, an autosomal recessive disease in humans, there exists an increased sensitivity to damage by x-ray. Patients with Fanconi’s anemia have defective repair of cross-linking damage.
iii. All these clinical syndromes are associated with increased frequency of cancer.