DNA REPLICATION

Group 321/08/2012

DNA Replication

•1: Initiation•2: Leading Strand•3: Lagging Strand•4: Termination•5: Enzymes•6: Regulation

Initiation

Initiation

• Initiation starts when 1 double-stranded DNA molecule produces two identical copies of the molecule

• DNA replication begins at specific locations in the genome called origins

• Origins are targeted by proteins that separate the 2 strands and initiate the DNA synthesis

Initiation

• Origins contain DNA sequences recognised by replication initiator proteins, e.g. DnaA in E.Coli, origin recognition complex in yeast

• These initiators recruit other proteins to separate the strands and initiate replication forks

• These initiators recruit other proteins and form pre-replication complex

• This separates the DNA strands at the origin and forms a bubble

Initiation

• Origin tends to be “A, T-rich” to assist this process because A,T pairs have only 2 H bonds

• In general, strands rich in these nucleotides are easy to separate because lesser number of H bonds require less energy to break them

• All known DNA replication systems require a free 3’ OH group before synthesis can be initiated

Leading Strand

Leading Strand• The leading strand is the template strand of the DNA double

helix so that the replication fork moves along it in the 3' to 5' direction.

• This allows the newly synthesized strand complementary to the original strand to be synthesized 5' to 3' in the same direction as the movement of the replication fork.

• No known DNA polymerase is able to begin a new chain. DNA polymerase can add a nucleotide onto only a preexisting 3'-OH group, and, therefore, needs a primer at which it can add the first nucleotide.

Leading Strand

• Primers consist of RNA and/or DNA bases. In DNA replication, the first two bases are always RNA, and are synthesized by another enzyme called primase.

• a: template, b: leading strand, c: lagging strand, d: replication fork, e: primer, f:Okazaki fragments

• On the leading strand, a polymerase "reads" the DNA and adds nucleotides to it continuously. This polymerase is DNA Polymerase III (DNA Pol III) in prokaryotes and presumably Pol ε in yeasts.

Leading Strand• In human cells the leading and lagging strands are synthesized by

Pol α and Pol δ within the nucleus and Pol γ in the mitochondria. Pol ε can substitute for Pol δ in special circumstances.

• As seen in, the nucleotides are added in the 5' to 3' direction. Triggered by RNA primase which adds the first nucleotide to the nascent chain, the DNA polymerase simply sits near the replication fork, moving as the fork does, adding nucleotides one after the other, preserving the proper anti-parallel orientation.

• This sort of replication, since it involves one nucleotide being placed right after another in a series, is called continuous.

Lagging Strand

Lagging Strand

• The lagging strand is the strand of the template DNA that is oriented so that the replication fork moves along in 5’-3’ direction.

• Because of its orientation, opposite to the working orientation of DNA polymerase 3 which moves on a template in 3’-5’ manner, replication of the lagging strand is more complicated than that of the leading strand.

• On the lagging strand, primase reads the DNA and adds RNA to it in short, separated fragments. In eukaryotes, primase is intrinsic to Pol alpha(enzyme).

Lagging Strand

• Pol delta or DNA polymerase 3(enzyme)lengthens the primed segment, forming okazaki fragments

• They were originally discovered in 1966 by Kiwako Sakabe and Reiji Okazaki in their research on DNA replication of Escherichia coli.

• They were further investigated by them and their colleagues through their research including the study on bacteriophage DNA replication in Escherichia coli

• It is also involved in primer(starting point ofDNA synthesis) removal in eukaryotes.

Lagging Strand

• In prokaryotes, DNA polymerase 1 reads the fragments ,removes the RNA using its flap endonuclease(enzyme) domain(RNA primers are removed by 5’-3’ exonuclease activity of polymerase 1 and replaces the RNA nucleotides with DNA nucleotides. This is because DNA and RNA have slightly different kinds of nucleotides.)

• In the end , DNA ligase joins the fragments together.

Okazaki Fragments• Okazaki fragments are short, newly synthesized DNA fragments

that are formed on the lagging template strand during DNA replication.

• They are complementary to the lagging template strand, together forming short double-stranded DNA sections.

• Okazaki fragments are between 1,000 to 2,000 nucleotides long in Escherichia coli and are between 100 to 200 nucleotides long in eukaryotes.

• They are separated by ~10-nucleotide RNA primers and are unligated until RNA primers are removed, followed by enzyme ligase connecting (ligating) the two Okazaki fragments into one continuous newly synthesized complementary strand.

Termination

TERMINATION

There are multiple sites of replication

And So are the Termination Sites

Termination involves 2 basic steps

• Search for the termination sites on the DNA strand

• Binding of the protein which will terminate the replication forks

Luckily the name of the protein is Ter Protein.

But before termination , there is a problem with the complete replication of the DNA strand• Eukaryotic cells have linear chromosomes,

and experiments suggest that it is not possible to replicate the strand of DNA at the Telomere region.

• So Do we lose a part of DNA in each cell division ???

Telomerase : Our saviour

Few Facts

• Telomere/Telemorasae are very closely related to aging.

• But unwanted activation of Telomerase in Body cells can lead to Cancer.

Enzymes

Enzymes Involved

• 1.DNA helicase

• 2.DNA polymerase

• 3.DNA ligase

• 4.DNA topoisomerase

• Enzyme helicase opens the helical DNA and extends the melted region

• enzyme topoisomerase releases the strain due to over winding

DNA ligase helps to bind newly formed DNA fragments 

DNA polymerase forms new DNA

Regulation

Regulation In Eukaryotes• In Eukaryotes, regulation of DNA replication is done through the

cell cycle

Regulation In Eukaryotes• In Eukaryotes, regulation of DNA replication is done through the cell

cycle• As the cell grows and divides, it goes through stages in the cell

cycle; DNA replication occurs during the S phase (synthesis phase)

Regulation In Eukaryotes• In Eukaryotes, regulation of DNA replication is done through the cell

cycle• As the cell grows and divides, it goes through stages in the cell

cycle; DNA replication occurs during the S phase (synthesis phase)• The progress of the eukaryotic cell through the cycle is controlled by

cell cycle checkpoints

Regulation In Eukaryotes• In Eukaryotes, regulation of DNA replication is done through the cell

cycle• As the cell grows and divides, it goes through stages in the cell

cycle; DNA replication occurs during the S phase (synthesis phase)• The progress of the eukaryotic cell through the cycle is controlled by

cell cycle checkpoints• The G1/S checkpoint (or restriction checkpoint) regulates whether

eukaryotic cells enter the process of DNA replication and, further, division

Regulation In Eukaryotes• In Eukaryotes, regulation of DNA replication is done through the

cell cycle• As the cell grows and divides, it goes through stages in the cell

cycle; DNA replication occurs during the S phase (synthesis phase)• The progress of the eukaryotic cell through the cycle is controlled

by cell cycle checkpoints• The G1/S checkpoint (or restriction checkpoint) regulates

whether eukaryotic cells enter the process of DNA replication and, further, division

• Replication of chloroplast and mitochondrial genomes occurs independent of the cell cycle, through the process of D-Loop Replication

D Loop Replication

• D-loop replication is a process by which chloroplasts and mitochondria replicate their genetic material

D Loop Replication

• D-loop replication is a process by which chloroplasts and mitochondria replicate their genetic material

• Many chloroplasts and mitochondria have a single circular chromosome like bacteria instead of the linear chromosomes found in eukaryotes

D Loop Replication

• D-loop replication is a process by which chloroplasts and mitochondria replicate their genetic material

• Many chloroplasts and mitochondria have a single circular chromosome like bacteria instead of the linear chromosomes found in eukaryotes

• In many organisms, one strand of DNA in the plastid comprises heavier nucleotides(more purines:A and G). This strand is called the H (heavy) strand.

D Loop Replication

• D-loop replication is a process by which chloroplasts and mitochondria replicate their genetic material

• Many chloroplasts and mitochondria have a single circular chromosome like bacteria instead of the linear chromosomes found in eukaryotes

• In many organisms, one strand of DNA in the plastid comprises heavier nucleotides(more purines:A and G). This strand is called the H (heavy) strand.

• Replication begins with replication of the heavy strand starting at the D-loop. An origin of replication opens, and the heavy strand is replicated in one direction. After heavy strand replication has continued for some time, a new light strand is also synthesized

Diagram of D-Loop Replication

Regulation in Bacteria

• Most bacteria do not go through a well-defined cell cycle but instead continuously copy their DNA

Regulation in Bacteria

• Most bacteria do not go through a well-defined cell cycle but instead continuously copy their DNA

• In E. coli DNA replication is regulated through various mechanisms:

− Because E. coli methylates GATC DNA sequences, DNA synthesis results in hemimethylated sequences. This hemimethylated DNA is recognized by the protein SeqA, which binds and sequesters the origin sequence

− ATP competes with ADP to bind to DnaA, and the DnaA-ATP complex is able to initiate replication

− A certain number of DnaA proteins are also required for DNA replication

THANK YOU