Objectives
DNA Replication and its importance
Different models of replication
Meselson and Stahl Experiment
Types of DNA Replication
Various Enzymes/Proteins involve in DNA replication
Mechanism of DNA Replication
What is DNA Replication?
DNA replication is the process by which a double-stranded DNA molecule is copied to produce two identical DNA molecules
Why DNA replication is essential?
Cell Division (Mitosis/Meiosis)
Reproduction
Synthesis of RNA and Protein
Two parental DNA strands are copied as such
That is, one daughter molecule contains both parental DNA strands, and the other daughter molecule contains DNA •strands of all newly- synthesized material
The two parental DNA strands separate and each
Separated strands then serves as a template for the synthesis of a new DNA strand
The result is two DNA double helices, both of which consist of one parental and one new strand
The parental double helix is broken into double-stranded DNA segments
These fragments act as templates for the synthesis of new double helix molecules. The segments then reassemble into complete DNA double helices
A. Semiconservative model
B. Conservative model
C. Dispersive model
They cultured a bacteria in a N15 medium
They then shifted the bacteria to a N14 medium
DNA was isolated at different times correspondig to replication cycle 0, 1 and 2
Then seprated the DNA with CsCI density gradient centrifugation
Theta replication
Rolling-circle replication
Linear eukaryotic replication
A template consisting of single-stranded DNA,
Raw materials (substrates i.e. dNTPs) to be assembled into a new nucleotide strand, and
Enzymes and other proteins that “read” the template and assemble the substrates into a DNA molecule.
DNA Helicase
Primase (RNA Polymerase)
Single Stranded Binding Proteins
DNA Polymerase
Topoisomerase (DNA Gyrase)
DNA Ligase
DNA strands always elongate in the 5’to 3’ direction (5’$\longrightarrow$ 3’).
Why? (5’$\longrightarrow$ 3’) Direction
Bacterial DNA replication involves -
Initiation
Elongation
Termination
E. coli has a single replication origin (oriC)
Initiator proteins bind to oric and cause a short section of DNA to unwind
This unwinding allows helicase to separate the double strand of DNA
Topoisomerases control the supercoiling of DNA
It reduces torque/tension by making a double-stranded break in one segment of the DNA helix, passing another segment of the helix through the break, and then resealing the broken ends of the DNA
Because of this requirement, DNA polymerases cannot initiate DNA synthesis on a bare template; rather, they require a primer-an existing 3-OH group-to get started.
How, then, does DNA synthesis begin?
DNA polymerases elongate the polynucleotide strand by catalyzing DNA polymerization
There are five different DNA polymerases
Two of them, DNA polymerase I and DNA polymerase III, carry out DNA synthesis associated with replication; the other three have specialized functions in DNA repair
Its 5’ $\rightarrow$ 3’ polymerase activity allows it to add new nucleotides in the 5’$\rightarrow$ 3’ direction
Its 3’$\rightarrow$ 5’ exonuclease activity allows it to remove nucleotides in the 3’$\rightarrow$ 5’ direction, enabling it to correct errors
The removal and replacement of primers appear to constitute the main function of DNA polymerase I
DNA polymerases II, IV and V function in DNA repair
Synthesize any sequence specified by the template strand
Synthesize in the 5’ $\rarr$ 3’ direction by adding nucleotides to a ‘3’-OH group
Use dNTPs to synthesize new DNA
Require a primer to initiate synthesis
Catalyze the formation of a phosphodiester bond by joining the 5’ phosphate group of the incoming nucleotide to the ‘3’-OH group of the preceding nucleotide on the growing strand
Produce newly synthesized strands that are complementary and antiparallel to the template strands
Termination In some DNA molecules, replication is terminated whenever two replication forks meet
In others, specific termination sequences block further replication
A termination protein, called Tus in E. coli, binds to these sequences
Helicase to unwind the DNA
Single-strand-binding proteins to keep the nucleotide strands separate long enough to allow replication
The topoisomerase, gyrase to remove strain ahead of the replication fork
Primase (RNA Polymerase) to synthesize primers with a ‘3’-OH group at the beginning of each DNA fragment
DNA polymerase to synthesize the leading and lagging nucleotide strands and Also removes primers
DNA Ligase seal the nick
The most obvious differences
Eukaryotes have -
Multiple replication origins in their chromosomes
More types of DNA polymerases, with different functions
Nucleosome assembly immediately following DNA replication
In Yeast, Autonomously replicating sequences (ARSs) enabled any DNA to which they were attached to replicate
A multiprotein complex, the origin recognition complex (ORC), binds to the ARS and unwinds the DNA in this region
DNA polymerase alpha, which contains primase activity, initiates nuclear DNA synthesis by synthesizing an RNA primer, followed by a short string of DNA nucleotides
After DNA polymerase alpha has laid down from 30 to 40 nucleotides, DNA polymerase delta completes replication on the leading and lagging strands
A fundamental difference between eukaryotic and bacterial replication arises because eukaryotic chromosomes are linear and thus have ends
The ends of chromosomes-the telomeres-possess several unique features, one of which is the presence of many copies of a short repeated sequence
End of DNA is replicated with the help of telomerase enzyme
Replication is always semiconservative
Replication begins at sequences called origins
DNA synthesis is initiated by short segments of RNA called primers
The elongation of DNA strands is always in the 5’ 3’direction
New DNA is synthesized from dNTPs
Replication is continuous on the leading strand and discontinuous on the lagging strand
New nucleotide strands are made complementary and antiparallel to their template strands
Replication takes place at very high rates and is accurate, because of precise nucleotide selection, proofreading, and repair mechanisms
Why DNA replication is required?
Which enzyme is responsible for polymerization of DNA?
What is the direction of DNA replication and why?
What are okazaki fragments?