REPLICATION OF DNA – ITS PROCESS, STEPS AND ENZYMES INVOLVED

DNA replication is the process by which two identical copies of DNA are produced from parent DNA molecule before the cell divides so that each daughter cell gets a complete set of DNA after cell division. It is semi-conservative in nature as newly formed DNA has one original DNA strand of parent DNA and one newly synthesized strand. Also, the two copies of replicated DNA are identical.

It occurs during S phase of cell cycle. The primary function of DNA replication is provision of progeny between the genetic information possessed by the parent and offspring.

Prokaryotes like bacteria have circular DNA which replicate fast in cytoplasm using a single origin of replication (Ori C). In eukaryotic cells, with linear DNA, replication is rather slow occurring in the nucleus during S phase of cell cycle using multiple origin sites.

STEPS OF DNA REPLICATION

• Identification of origin of replication.
• Unwinding of double stranded DNA.
• Formation of replication fork and synthesis of RNA primer.
• Initiation of DNA synthesis and elongation.
• Termination.
• Reconstitution of chromatin structure.

ENZYMES AND PROTEINS INVOLVED IN DNA REPLICATION

• HELICASE – Causes unwinding of DNA by breaking the hydrogen bond between DNA bases.
• DNA POLYMERASE – Results in deoxynucleotide polymerization to build a new strand of DNA.
• TOPOISOMERASE – Relieves torsional strain that results from helicase – induced unwinding.
• DNA PRIMASE – Initiates synthesis of RNA primers.
• DNA LIGASE – It helps to seal the DNA fragments together.
• SINGLE STRAND BINDING PROTEIN – Prevents premature reannealing of DNA strands.

Let us now read the steps of DNA replication.

IDENTIFICATION OF ORIGIN OF REPLICATION

DNA replication starts at the origin. Prokaryotes have only one origin while eukaryotes have multiple origins. This site is identified by certain DNA sequences. At the origin of replication ( ori ), there is an association of sequence specific ds DNA binding proteins with a series of direct repeat DNA sequences. This results in local denaturation and unwinding of an adjacent A+T rich regions of DNA.

UNWINDING OF DNA

The two strands of double stranded DNA are separated by enzyme helicase which causes processive unwinding of DNA by breaking hydrogen bond between the complementary DNA bases. Single stranded DNA binding proteins ( SSBs ) keep the two strands separated.

FORMATION OF REPLICATION FORK

A replication fork consists of four components that form in the following sequence :

• DNA helicase unwinds a short segment of ds parent DNA.
• Primase initiates synthesis of an RNA molecule that act as primers for DNA synthesis.
• DNA polymerase initiates synthesis of nascent, daughter DNA strand.
• SSBs bind to ssDNA and prevent premature reannealing of two strands.

INITIATION AND ELONGATION OF DNA SYNTHESIS

The initiation of DNA synthesis requires priming by a short length of RNA, about 10 – 200 nucleotides long. In eukaryotes, DNA polymerase A synthesizes RNA primers. In prokaryotes, this step is catalyzed by dna G.

The priming process involves nucleophilic attack by the 3′-hydroxyl group of the RNA primer on the phosphate of the first entering deoxynucleoside triphosphate causing the splitting off of pyrophosphate. This reaction is catalyzed by DNA polymerase III in prokaryotes and DNA polymerase delta and epsilon in eukaryotes. This reaction then goes on to proceed on subsequently added deoxyribonucleoside triphosphates.

DNA polymerase enzyme ( DNA polymerase III in prokaryotes and dna E in eukaryotes ) binds to template DNA. It causes polymerization of deoxynucleotides thus synthesizing new strand of DNA.

DNA polymerase can synthesize DNA only in 5′ – 3′ direction. The DNA strands are antiparallel (one strand runs in 5′-3′ direction and other in 3′-5′ direction). Therefore, DNA polymerase functions asymmetrically on both strands. On the template strand that runs in 3′-5′ direction, known as leading strand (forward strand), DNA is synthesized continuously in 5′-3′ direction. On the other hand, on the strand running in 5′-3′ direction, known as the lagging strand (retrograde strand), DNA is synthesized discontinuously. DNA polymerase epsilon causes leading strand synthesis and DNA polymerase delta is responsible for lagging strand synthesis.

DNA is synthesized in short fragments, called Okazaki fragments (150-250 nucleotides long in eukaryotes and 1000-2000 nucleotides long in prokaryotes) on the lagging strand, again in 5′-3′ direction as helicase unwinds the lagging strand (unwinding occurs in 5′-3′ direction on lagging strand). The gaps between Okazaki fragments are filled with complementary base paired deoxynucleotide. DNA ligase then joins the Okazaki fragments.

TERMINATION OF DNA REPLICATION

When the two replication forks meet, the process of DNA replication is terminated.

RECONSTITUTION OF CHROMATIN STRUCTURE

Chromatin structure must be re-formed after DNA replication in eukaryotes. Histone octamers are randomly distributed to each arm of replication fork and newly replicated DNA is rapidly assembled into nucleosomes. These reactions are facilitated by histone chaperone proteins.

In eukaryotic cell, the rate of polymerization is slower than in prokaryotes due to interference by nucleosomes in eukaryotes.

The proofreading function identifies and corrects copying errors during DNA replication. DNA polymerase I and II are mostly involved in proofreading and DNA repair.