DNA Replication-Lecture-1

DNA Replication: the process of DNA Synthesis

Purpose of replication

• Basis for inheritance
• The fundamental process occurring in all cells for copying DNA to transfer the genetic information to daughter cells
• Each cell must replicate its DNA before division.

Salient features of DNA replication

1) Semi conservative

• Parental strands are not degraded
• Base pairing allows each strand to serve as a template for a new strand
• New duplex is 1/2 parent template & 1/2 new DNA (figure-1)

Figure-1- Semiconservative mode of replication

2) Semi discontinuous- Leading & Lagging strands

• Leading strand – continuous synthesis
• Lagging strand – Okazaki fragments joined by ligases (figure-2)

Figure-2- Semi- discontinuous type of replication

 3) The energy of Replication- The nucleotides arrive as nucleoside triphosphates with their own energy source for bonding

• DNA base, sugar with PPP
• P-P-P = energy for bonding 
• Mono phosphate form is bonded by enzyme DNA polymerase III, a pyrophosphate is released, which is further broken down, the energy released is used for polymerization.

 GTP———————-> GMP +PPi

4)  Template reading and direction of polymerization         

• Each DNA strand serves as a template to guide the synthesis of the complementary strand.
• The template is read in the 3′-5′ direction while polymerization takes place in the 5′-3′ direction.

5) Primer is needed

The DNA polymerase can only add nucleotides to 3′ end of a growing DNA strand needs a “starter” nucleotide to make a bond

• Primer serves as a starter sequence for DNA polymerase III
• RNA primer is synthesized by Primase
• RNA Primer has a free 3’OH group to which the first Nucleotide is bound (figure-3)
• Only one RNA Primer-required for the leading strand
• RNA Primers for the lagging strand depend on the number of “OKAZAKI FRAGMENTS”

Figure 3- The deoxyribonucleotides are attached to the 3’OH group of the primer by 3’-5’ phosphodiester linkage.

 DNA Replication (Prokaryotes) Steps

• Identification of the origins of replication
• Unwinding (denaturation) of dsDNA to provide an ssDNA template
• Formation of the replication fork
• Initiation of DNA synthesis and elongation
• Primer removal and ligation of the newly synthesized DNA segments
• Termination of replication

Components of Replication

Enzymes

• DNA polymerases- Deoxynucleotide polymerization (I, II, and III)
• Helicase -Processive unwinding of DNA
• Topoisomerases (I and II) relieve the torsional strain that results from helicase-induced unwinding
•  RNA primase initiates the synthesis of RNA primers
•  DNA ligase-seals the single strand nick between the nascent chain and Okazaki fragments on the lagging strand

Proteins

• dna A protein for identification of the origin of replication
• Single-strand binding proteins prevent premature reannealing of dsDNA and to protect from attack by nucleases
• Deoxyribonucleotides for polymerization

Step-1-  Identification of Origin of Replication

• At the origin of replication (ori), there is an association of sequence-specific dsDNA-binding proteins with a series of DNA sequences.
• In E Coli, the oriC is bound by the protein dnaA (figure-4)
• A complex is formed consisting of 150–250 bp of DNA and multimers of the DNA-binding protein.
• This leads to the local denaturation and unwinding of an adjacent A+T-rich region of DNA.

Step-2- Unwinding of double-stranded DNA to provide a single-stranded template

• The interaction of proteins with ori defines the start site of replication and provides a short region of ssDNA essential for initiation of synthesis of the nascent DNA strand.
• DNA Helicase allows for the processive unwinding of DNA.
• Single-stranded DNA-binding proteins (SSBs) stabilize this complex.
• Replication occurs in both directions along the length of DNA and both strands are replicated simultaneously.
• This replication process generates “replication bubbles” (figure-4)
• A further unwinding of DNA creates a replication fork.

Figure-4- Identification of the origin of replication and formation of a replication bubble.

Step-3 Formation of Replication Fork-

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

• DNA helicase unwinds a short segment of the parental duplex DNA
• A primase initiates the synthesis of an RNA molecule that is essential for priming DNA synthesis;
• DNA polymerase initiates nascent, daughter strand synthesis; and
• SSBs bind to ssDNA and prevent premature reannealing of ssDNA to dsDNA (figure-5)

 

Figure-5 – components and the processes involved at the replication fork.