DNA replication

DNA replication:

The flow of the biological information begins with DNA replication. DNA replication is necessary for cells to divide, whether to reproduce new organisms as in unicellular organisms or to produce new cells as part of multi-cellular organisms. There is three hypothesis proposed for nature of DNA replication.

1. Conservative
2. Semi-conservative
3. Dispersive

Stahi and meselson proved the semiconservative nature of DNA replication in stahi and meselson experiment are given below:

1. They grew E. Coli cells for many generations in medium containing 15NH₄Cl (ammonium chloride) as a source of nitrogen.
2. They transfer E. Coli cells in medium containing NH₄Cl where nitrogen was 14N.
3. They isolate the DNA and centrifuged in CsCl (cesium chloride) gradient. This time, they found that DNA was precipitated in the hybrid zone between ¹⁵N and ¹⁴
4. Again, they transfer E. Coli cells in ¹⁴N medium repeated the process. This time, they found that two bands of DNA present in hybrid zone and light zone i.e¹⁴
5. In this way, they demonstrated the DNA replication is semi-conservative.

Templates and enzymes:

DNA exists in cells as a double helix with the complementary base pairing. If the DNA double helix is opened up a new strand can be synthesized as the complement of each parental strands. The replication is semi-conservative meaning that the two resulting double helixes consist of one newly made strand and one parental strand.

The precursor of each nucleotide in the chain is a deoxyribonucleoside –s’ triphosphate, from which two terminal phosphates are removed and internal phosphate is bonded covalently to the deoxyribose of growing chain. The addition of the nucleotide to the growing chain requires the presence of free hydroxyl group which is available only at the 3’ end of the molecule because of this DNA replication always proceeds from the 5’end to the 3’ end.

Major enzymes involved in DNA replication in bacteria.

DNA polymerase and primase:

Enzymes that catalyze the addition of deoxynucleotides are called DNA polymerase. There are 5 different DNA polymerase in E. Coli called DNA polymerase I,II,III,IV and V. DNA polymerase III is the major polymerizing enzyme.

To start a new chain a primer , a nucleic acid molecule to which DNA polymerase can add, the first deoxyribonucleotide, is required. In most cases, the primer is the short stretch of RNA around 11-12 nucleotides synthesized by enzymes called primase. Continued extension of the molecule occurs as DNA rather than RNA. The primer will eventually be removed and replaced with DNA.

1. Initiation of DNA synthesis:

Before DNA polymerase can synthesize new DNA the pre-existing double helix most is unwound to expose the template strands. The zone of unwinding DNA where replication occurs is known as the replication fork. An enzyme known as DNA helicase unwinds the double helix and exposes a short single-stranded region. The energy required by helicase for unwinding comes from the hydrolysis of ATP. Helicase moves along the helix and separates the strand just in advance of the replication fork. The single strand binding protein binds the single-stranded DNA and prevents from annealing.

In prokaryotes, there is a single location on the chromosome, the origin of replication, where DNA synthesis is initiated. This consist of a specific DNA sequence of about 250 bases, that is recognized by the particular protein called DNA. Initiation of DNA replication begins on the two single strand. As replication proceeds, the replication fork appears to move along the DNA.

Leading and lagging strands:

On the strand growing from the 5’ PO₄^—to the 3’-OH called the leading strand, DNA synthesis occurs continuously because there is always a tree 3’-OH group at the replication fork to which a new nucleotide can be added. But on the opposite strand caller the lagging strand, DNA synthesis occurs discontinuously because there is no 3’-OH at the replication fork to which a new nucleotide can attack, 3’-OH group is located at the opposite end away the replication fork. Therefore, on the lagging strand, RNA primers must be synthesized multiple times to provide free 3’-OH groups. As a result, the lagging strand is made in short segments, called Okazaki fragments.

2. Elongation:synthesis of new DNA strand:

After synthesizing the RNA primer, primase is required by DNA polymerase III. Each polymerase is held in the DNA by a sliding clamp, which encircles and slides along the single template strands of DNA. Consequently, the replication fork contains two polymerase core enzymes and two sliding clamps. After assembly, the DNA polymerase then adds deoxyribonucleotides. DNA polymerase I remove the RNA primer and replaces with DNA. DNA ligase joins the strands of DNA.

In e. Coli and probably in all prokaryotes with chromosome, replication is bidirectional from the origin of replication. There are thus 2 replication forks on each chromosomes moving in opposite direction. In circular DNA bidirectional replication leads to the formation of characteristics structure called theta structures. Bidirectional DNA synthesis along circular chromosomes allows DNA to replicate as rapidly as possible.

Proofreading and termination

DNA replicates with a low error rate. Never the less when errors do occur, a backup mechanism exist to detect and correct them.

Proofreading during DNA replication: Errors in DNA replication introduce mutations.Mutation rates in cells are low between 10^-8 to 10^-11 errors per base inserted.this accurately is partly possible because DNA polymerase gets two chances to incorporate the correct base at the given site. The first chance follows the insertion of the complementary base opposite the template strand by polymerase II according to the base pairing rules. The second chance depends on upon the second enzymatic activity of both polys I and poly III called proofreading.

Termination of DNA replication: On the opposite of the circular chromosome from the origin is a site called terminus of replication. Here two replication forks collide as the new circles of DNA are completed.In the terminus region, there are several DNA sequences.

References

Arvind, Keshari K. and Kamal K Adhikari. A Textbook of Biology. Vidyarthi Pustak Bhander.

Michael J.Pleczar JR, Chan E.C.S. and Noel R. Krieg. Microbiology. Tata Mc GrawHill, 1993.

Powar. and Daginawala. General Microbiology.

Rangaswami and Bagyaraj D.J. Agricultural Microbiology.