REPLICATION

REPLICATION

Semi-conservative replication of Chromosomes in eukaryotes:

Autoradiography experiment in Vicia faba, by J.H. Taylor and his co-workers for the study of duplicating chromosomes in the root tip cells, were first published in 1957. They reported that

DNA in all the organisms has the inherent capacity of self-replication. The mechanism of DNA replication is so precise that all the cells derived from a zygote contain exactly similar DNA both in terms of quality and quantity. The replication takes place in interphase after every cell division.

Theoretically, there may be following three possible modes of DNA replication:

Dispersive Method

Conservative Method

Semiconservative Method

The semiconservative mode is the most accepted of all.

Semiconservative Method: - During replication, the two strands of the DNA molecule uncoil with the help of some proteins and enzymes. The unpaired bases in the single-stranded regions of the two strands bind with their complementary bases in the single-stranded regions of the two stands bind with their complementary bases present in the cytoplasm in the form of nucleotides. These nucleotides become joined by phosphodiester linkages generating complementary strands on the old ones. This provides for an almost error-free, high fidelity replication of DNA.

Detailed mechanism of semiconservative mode of DNA replication was given by Kornberg. He proposed that following enzymes are important for functional replication of DNA.

1. Nucleases
2. Unwinding Proteins
3. DNA Polymerases
4. DNA Ligases
5. RNA primer
6. Primases or RNA polymerases.

Out of the above six enzymes/proteins, the three; nucleases, RNA polymerases and DNA polymerases are known as “Kornberg Enzyme”.

1.  NUCLEASES

These are the enzymes which digest or breakdown nucleic acid molecules. These attack on phosphodiester bonds of the nucleic acid backbone and release nucleotides through hydrolysis. On the basis of their mode of function, nucleases may be classified into following two:

(i)  Exonucleases

(ii)  Endonucleases.

Exonucleases function on phosphodiester bonds of the DNA at both the terminus. The endonucleases, on the other hand attack the phosphodiester bonds at intercalary regions of the DNA breaking it into as many parts as the site of function.

2.   UNWINDING PROTEINS.

Unwinding proteins are those proteins or enzymes, which uncoil the DNA helix and separate the two DNA strands by breaking hydrogen bonds between them. Due to this function, these are known as unwinding proteins.

Due to separation, the two strands form a ‘Y’ or fork-like structure, which is known as replication fork. Usually, two types of unwinding proteins have been recognized:

(i)  DNA helicases: These enzymes or proteins uncoil the helix of DNA which may now appear as a ladder.

(ii)  DNA gyrases: These enzymes break down the hydrogen bonds (A=T, C=G) between two strands of a DNA molecule. E.g., DNA topoisomerase.

3.  DNA Polymerases or Replicase Enzyme

DNA polymerases are the first enzymes suggested to be implicated in DNA replication. It mainly functions in the polymerization of nucleotides on the DNA template producing thereby the polynucleotide chain.

In eukaryotic cells, these three enzymes have been named as  DNA polymerase, β-DNA polymerase and -DNA polymerase. Due to their role in DNA replication, DNA polymerases are also known as DNA replicates.

(1)   DNA POLYMERASE-I: This enzyme was first discovered by Arthur Kornberg in E.coli. After the name of the scienctist, DNA polymerase-I is also known as ‘Kornberg enzyme’ or ‘Kornberg polymerase’. It is the most extensively studied DNA polymerase studied in DNA replication machinery of E.coli. Nowadays, it is believed that this enzyme is not responsible for DNA replication and it mainly function in DNA repair.

Structurally, a molecule of DNA polymerase-I consists of a single polypeptide chain having the molecular wt. 109,000. An atom of Zn is associated with each molecule of this enzyme.

Following sites have been reported to be present on its surface:

(a)  Template Site: it is occupied the DNA strand.

(b)  Primer Site: The site at which the growth of DNA chain occurs.

(c)  Nucleotide triphosphate site: The site where the incoming nucleotide triphosphate is received.

(d) Primer terminus site: This site, which is used for removing any mismatched nucleotide at the end of growing chain.

(e) Site for 5’­à 3’ cleavage: The site which is used for removing any strand coming in the path of growing primer. DNA polymerase-I function in following activities within cells.

(i)  5’à 3’ polymerization activity: Attachment of nucleotides with each other by the activity of DNA polymerase forming a polynucleotide chain is called polymerization. The rate of polymerization in E.coli at 37⁰ C has been noted to be 1000 nucleotides per minute. It occurs in 5’à 3’ direction. It forms small DNA segments through polymerization, which is used in repair mechanism.

(ii)  3’à 5’ Exonuclease Activity: The activity of DNA polymerase-I is performed to remove any nucleotide which mispairs during elongation of growing strand.

(iii) 5’à 3’ Exonuclease Activity: This activity is performed by this enzyme to remove any DNA segment which comes as an obstruction in the way of growing DNA Strand.

(iv) Removal of Thymine Dimmers: DNA polymerase-I function in the removal of thymine dimers from the DNA strand. Such thymine dimmers are produced to UV irradiation. After removal of diers, it also fills the gap, formed due to excision.

(2) DNA Polymerase-II: This enzyme has also been isolated from E.coli and has the molecular wt. 90,000. Polymerization rate DNA polymerase-II is much slower than polymerase-I. It is only 50 nucleotides per minute in E.coli, it has 3’à 5’ exonuclease activity but lacks 5’à3’ exonuclease activity, unlike polymerase-I.

(3) DNA- Polymerase-II I: This is the main DNA polymerase involved in DNA replication. This polymerase enzyme was originally discovered in a lethal mutant of E.coli having mutation at the dnaE locus. The enzyme has a higher affinity for nucleotides than polymerase-I and II have. The rate of polymerization by polymerase-III is approximately 10-15 times higher than the polymerase-I.

Structurally, DNA polymerase molecule consists of two polypeptide chains each having the molecular wt. of 90,000. This dimeric enzyme does not function unless it associates with two more chains of co-polymerase-III each having the molecular wt. of 77,000. The holoenzyme may be represented as α₂β₂ where α₂ polymerase-III and β₂ co-polymerase-III. ATP is also needed for the growth of the polynucleotide chain.

The enzyme DNA polymerase-III has 3’à5’ exonuclease activity and 5’à3’ polymerase activity.

4. DNA LIGASES

Through polymerase activity by DNA polymerase-III, polynucleotide chains is formed in the form of small fragments which are known as Okazaki fragments. In order to form a complete chain complementary to that of the template, ligation of okazaki fragments is essential. The ligation reaction is performed by RNA ligases.

5. RNA PRIMER

DNA replication really starts with the formation of a RNA fragment known as RNA primer. It is formed at the point of origin. The primer is formed throught polymerization activity by RNA polymerase. Due to this reason, RNA polymerase is also needed for functional replication of DNA.

6. PRIMASES

This is the group of enzymes, which are involved in RNA primer synthesis. RNA polymerase is an example of primases.

            Detailed molecular biological studies of the process of DNA replication now have revealed that the process is much complex and requires a multi-enzyme complex. About two dozens of enzymes are involved in this complex. The complex is known as ‘replisome’

STEPS OF DNA REPLICATION

Before studying the mechanism of DNA replication, we must be familiar with following terms:

1. Replicon:  A replicon is the unit of DNA in which individual act of replication takes place. It has capacity of DNA replication independent of other segments. Therefore, each replicon has its own origin and terminus, at which DNA replication stops.

2. Origin: This is the sequence of a replicon which supports initiation of DNA replication and also regulates the frequency of replication initiation. A general feature of origin is that it is A=T rich. An origin in E.coli, oric  has been identified to 250 base pairs long.

3. Terminus: In most of prokaryotes, replicons has a specific site at the extreme downstream of the strands. This site stops replication fork movement and thereby terminates DNA replication.

In order to understand the exact mechanism of DNA replication. The process must be studied in stepwise manner.

The overall process is completed in following steps:

(1) Before the start of DNA replication and formation of origin point, the enzyme DNA helicase associates with the site of DNA. Its molecules unwind the two strands of the DNA. Another enzyme, DNA gyrase or DNA topoisomerase breaks the hydrogen bonds between the two strands and separate them from each other forming a ‘Y’ shaped replication fork.

(2) The two strands of a DNA molecule separated in the way explained in the first step function as template. It should be noted here that template is the single strand of DNA on which polymerization of nucleotides forming a new strands takes place.

In eukaryotes, evidences for bi-directional DNA replication are available. DNA replication starts at many points each of which start as a loop and can be seen as expanding bubbles or eyes in electron micrograph.

The number of eyes or bubbles indicates the number of replicons.

(2) Formation of RNA Primer: Before the actual replication of DNA starts at origin, a short fragment of RNA is synthesized with the help of RNA polymerase. This RNA fragment is called RNA primer. It is believed that it provides safety to the new DNA strands, which is synthesized extending the RNA primer itself.

(3) Synthesis of Complementary Strand of DNA: DNA replication or synthesis of a new DNA strand complementary to the template is catalysed by the enzyme, DNA polymerase-III. It starts at the end RNA primer in 5’à3’ direction. The nucleotide sequence in the new strand is always complementary to the sequence of nucleotides in the template.

Some features of DNA replication are as follows:

(i) DNA Replication is Bi-Directional: Johan Cairsn on the basis of his experiments on atoradigraphy concluded that DNA synthesis starts at a fixed point on the chromosome and proceeds in one direction only. Subsequently, it was realized that Cairns results could be interpreted in terms of bi-directional replication also. On the basis of many other experiments it has convincingly been demonstrated that DNA replication begin at a unique site at origin and proceeds in both the direction on a strand. It takes place in the form of pieces called ‘Okazaki fragment which are joined with each other with the help of DNA ligases.

(ii) The two strands of the parent DNA at the point of replication fork or origin replicate together with each other.

(iii) Replication of 3’à5’ strand of DNA molecule is continuous and the new strand grows in 5’à3’direction. Replication in the second strand of the DNA molecule is discontinuous. Replication of this strand starts somewhat later than that of strand. Consequently, a given segment of 5à3’ strand always replicates i.e., the 3’à5’ strand. Therefore, the 3’à5’ strand of the parent DNA molecule is known as the leading strand while the 5’à3’ strand is termed as the lagging strand.

(iv) Formation of RNA primer takes place in the beginning of each and every okazaki fragments.

4. Termination of DNA Replication: The termination of DNA replication so signaled by specific sequences, the ter-elements. E.coli, the ter-element of R6 K plasmid has a 23 base pair sequence. This site functions as the binding site of Tus, a 36 K dal protein necessary for termination. This stops the replication fork movement and thereby stops DNA replication.

5. Removal of RNA Primer: After the whole DNA molecule is replicated on both the strands, the RNA primer on all the segments are removed or degraded with the help of DNA polymerase-I through its nuclease activity.

6. Synthesis of DNA Strand at the place of RNA Primer: in order to have replication of a complete DNA molecule, replication of the segment at the place of RNA primer is necessary. This process is performed with the help of DNA polymerase-I instead of polymerase-III through polymerization of deoxyribonucleotides. After DNA replication at the place of RNA primer, the replication process is completed.

7. Proof Reading and Repair Mechanism: The complementary base pairing during DNA replication is much accurate and precise, however, there are chances of error in this process of base pairing. It is mainly due to various physical and chemical forces involved in rate of error in E.coli are 5 x 10^-8 to 5 x 10^-10.

1. The proof reading of newly synthesized DNA strand is done by DNA polymerase-III through correction of mismatched base pairing. It deletes the wrong base and replaces it by a correct base. The process of proof reading also takes place in 5’à3’ direction. In E.coli, mutants defective in proof reading show an increase in mutation frequency by over 1000 times.