DNA Replication And It's Process In Prokaryotic DNA.

DNA Replication:

DNA replication is a complex enzymatic process where synthesis of new DNA takes place, using DNA template. The key enzyme involved in DNA replication is DNA-dependent DNA polymerase enzyme. DNA replication is semi-conservative in nature and is a bidirectional process.

DNA replication in the prokaryotes can be divided into various stages:

Step 1: Initiation of replication

- DNA polymerase enzyme cannot use double-stranded DNA as template DNA. The first step in DNA replication is unwinding or separation of the two complementary DNA strand.

- Initiation of unwinding starts at a specific nucleotide sequence called origin of replication.

- The specific nucleotide sequence is almost exclusively rich in A:T sequence.

- The process of unwinding involves the following proteins and enzymes:

a) DNA-A Protein:

DNA-A protein binds to the specific nucleotide sequence at the origin of replication. This ATP-requiring process causes the double stranded DNA to melt (separate) . The strands separate forming localized result of the region of single stranded DNA.

b) Single-Stranded Binding Protein (SSB Protein):

This protein not only keeps the two strands separated in the area of the replication origin, thus providing the single stranded DNA template required by DNA polymerase enzyme, but also protects the single stranded DNA from nuclease attack.

c) Helicase:

Helicase enzyme binds two single stranded DNA near replication fork and then moves into the neighboring double stranded region facing two strands apart.

The process needs ATP for breaking of H-bond.

Step 2: DNA Replication Fork

DNA polymerase cannot initiate synthesis of a complementary strand on a totally single stranded template rather they require RNA primer, a short double-stranded region consisting of RNA base paired to template DNAwith a free three prime (3') -OH group on the RNA strand. This -OH group serves as the acceptor of the first nucleotide by the action of DNA polymerase enzyme.

A specific RNA polymerase called primase synthesize the short stretch of RNA (approximately 10 nucleotides long) that are complementary and anti- parallel to the template DNA strand forming a hybrid duplex.

Step 3: Chain Elongation (Action Of DNA Polymerase)

DNA polymerase cannot initiate synthesis of complementary DNA strand on totally stranded DNA . Hence RNA polymerase enzyme provides face 3'-OH group.

DNA polymeraseIII elongates new DNA strand by adding deoxyribose nucleotides to the 3' end of growing chain which is initially RNA primer.

The sequence of nucleotides that are added are detected by the sequence of the template strand with which the incoming nucleotides are paired.

The new strand grows in 5' to 3' direction antiparallel to the template strand.

The nucleotide building blocks are deoxyribose nucleoside triphosphates. Pyrophosphate is released when each nucleoside is added to the growing chain. Pyrophosphate undergoes further hydrolysis to produce two inorganic phosphates. The energy released here is utilized for making phosphodiester bond.

Proof Reading Of Newly Synthesized DNA:

To ensure the replication fidelity, DNA polymeraseIII in addition to it's 5'-3' polymerase activity,it has a proofreading activity. As each nucleoside is added to the chain, DNA polymeraseIII checks to make certain that the added nucleoside is in fact correctly matched to its complementary base on the template DNA.

Step 4: Formation Of Leading And Lagging Strands

The DNA polymerase responsible for synthesizing new DNA strand is able to read the template strand 3'-5' direction and synthesizes new strand in 5'-3' direction. Hence beginning with the template strands, the two newly synthesized strands must grow in opposite direction i.e. one in 5'-3' direction towards the replication fork and other 5'-3' direction away from the replication fork.

Leading Strand:

The newly synthesized strand which is growing towards the replication fork is called leading strand.

Lagging Strand:

The newly synthesized strand which is growing away from the replication fork is called lagging strand.

Step 5: Excision Of RNA Primer

DNA polymeraseI removeRNA primer by its 5'-3' exonuclease activity and synthesizes equivalent DNA by it's 5'-3' polymerase activity. The final phosphodiester bond between the 5' phosphate group on the DNA chain synthesized by DNA polymeraseIII and the 3'-OH group on the DNA chain synthesized by DNA polymeraseI is catalyzed by DNA ligase enzyme.

Step 6: Solving The Problem Of Supercoiling

As the two strands of the double-stranded helix are separated, the appearance of super twisted or super coil arises in the region of DNA ahead of the replication. This problem is solved by an enzyme called DNA topoisomerase enzyme. There are two functions of DNA topoisomerase enzymes i.e. cutting and resealing.

Step 7: Termination Of DNA Replication

The detail about the process of termination of DNA replication is completely unknown. Certain DNA sequences called terminus sequences are found in diametrically opposite end of the origin of the replication where "T U S" protein (Terminus Utilization Substance) which slows down the movement of replication to be completed.

References:

(DL and MM)