Download - DNA Replication and Repair
DNA Replication and RepairFundamental Properties of Cells
Introduction All cells undergo DNA replication and
cell division in order to give rise to a new generation of cells
Mitosis- Division of the nucleus of a eukaryotic cell into two daughter nuclei with identical sets of chromosomes
Cytokinesis
Division of cell cytoplasm and organelles of a cell into two daughter cells
It is important that each daughter cell has an exact copy of the parent cell’s DNA
DNA Replication 1958- Matthew Meselson and Frank Stahl
devised a clever experiment that suggested that DNA replication is semiconservative
They grew E. coli in a medium using ammonium ions (NH4
+) as the source of nitrogen for DNA (as well as protein) synthesis
They worked with two isotopes of nitrogen 14N is the common isotope of nitrogen, but they
also used ammonium ions that were enriched for a rare heavy isotope of nitrogen, 15N.
Meselson & Stahl Experiment
Semiconservative Replication
Semiconservative replication is the process of replication in which each DNA molecule is composed of one parent strand and one newly synthesized strand (daughter strand)
Each daughter molecule receives one strand from the parent molecule plus one newly synthesized strand
Semiconservative Replication
The Process of DNA Replication Replication begins when proteins bind at a
specific site on the DNA known as the replication origin
In prokaryotes, the closed circular DNA has only one origin of replication
In eukaryotes, the linear DNA has multiple origins of replication
In both organisms, the two strands forming the DNA molecule cannot simply be pulled apart. Why?
Replication Origin The two strands of
the DNA molecule cannot be simply pulled apart because they are held together by hydrogen bonds that are twisted around each other to form a double helix.
DNA Helicase To expose a template strand, the two
parent DNA strands must be unravelled and kept separate.
DNA Helicase is a specific enzyme that unwinds the double helix by breaking the hydrogen bonds between the base pairs
SSBs DNA base pairs are
complementary to each other and have a natural tendency to anneal
Anneal: the pairing of complementary strands of DNA through hydrogen bonding
Single-stranded binding proteins (SSBs) are proteins that keep separated strands of DNA apart after DNA helicase has unwound them
SSBs bind to the exposed DNA single strands and block hydrogen bonding
DNA Gyrase The bacterial enzyme that relieves any
tension brought about by the unwinding of the DNA strands during replication
Gyrase works by cutting both strands of DNA, allowing them to swivel around one another, and then releasing the cut strands
Enzymes from the same family as gyrase have similar functions in eukaryotes
DNA Replication DNA cannot be fully unwound
because of its large size compared with the size of the cell
The diameter of a human cell is ~ 5 μm The length of DNA is ~1 cm, That is a 2000-fold difference As a region of the DNA unwinds,
replication begins in two directions from that origin
DNA Replication New complementary
strands are built as soon as an area of the DNA has been unwound
Replication fork: As the two strands of DNA are disrupted, the junction where they are still joined is called the replication fork
Replication Bubble In eukaryotes, more than one replication
fork may exist on a DNA molecule at once because of the multiple sites of origin
This results in hundreds or replication forks across a DNA strand and
allows for rapid replication of DNA When two replication forks are quite near
each other, a replication bubble forms
Origin of Replication
Replication Fork
Replication Bubble
Parental Strand
Daughter Strand
Replication fork
Replication bubbles
Origin of Replication
Eventually, the replication bubbles become continuous and the two new double-stranded daughter molecules are completely formed
Building the Complementary Strands
In Prokaryotes, DNA Polymerase I, II, and III are the three enzymes that function in DNA replication and repair
In Eukaryotes, five different types of DNA Polymerase are at work
Review: Nucleic Acid contains a chain of nucleotides covalently linked together via phosphodiester bonds to form a sugar-phosphate backbone with protruding nitrogenous bases.
Nucleotide A nucleotide consists of a nitrogenous base, a sugar, and a phosphate group
Nucleoside A nucleoside consists of a
nitrogenous base covalently attached
to a sugar (ribose or deoxyribose)
but without the phosphate group
Therefore, a nucleotide is a “nucleoside-mono-phosphate”
DNA Polymerase III DNA polyermerase III is the primary
enzyme responsible for replication. It's main function is to add the 5'
phosphate of a new nucleotide to and existing 3'-OH group.
Therefore, it synthesizes DNA in the 5' to 3' direction, i.e., it adds free deoxyribonucleoside triphosphates to a 3' end of an elongating strand
DNA Polymerase III Why does it add deoxyribonucleoside
triphosphates ? The high energy of the triphosphates is
used to form bonds between the nucleotides.
The two outermost phosphates are liberated, leaving the innermost group still attached.
Once the two outermost phosphates have been released, the nucleoside triphosphate has become a nucleotide
DNA Polymerase III This enzyme has its own limitations It can't begin a new daughter strand by
itself - it requires that there already be a 3'-OH end to add the next nucleotide to
Since DNA Polymerase III cannot initiate a new complementary DNA by itself, an RNA primer (10-60 base pairs of DNA) is annealed to the template strand
Primase The enzyme that builds RNA primers in a
5' to 3' direction since DNA polymerase III cannot begin initiating on its own
This primer provides the free 3'-OH needed by DNA polymerase III.
This initiation sequence is temporary and is later removed and replaced by DNA
Once in place, DNA Polymerase III can start elongation by adding free
to the complementary strand
Steps of DNA Replication The first major step for the DNA
Replication is the breaking of hydrogen bonds between bases of the two antiparallel strands.
The unwounding of the two strands is the starting point.
Helicase is the enzyme that splits the two strands.
Steps of DNA Replication The initiation point
where the splitting starts is called "origin of replication"
The structure that is created is known as Replication Fork
Steps of DNA Replication One of the most important steps
of DNA Replication is the binding of RNA Primase in the initiation point of the 3'-5' parent chain
RNA Primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3'-5' strand due to the hydrogen bonds between the bases.
nucleotides.
Steps of DNA Replication When DNA is being replicated, two
types of daughter strand are being produced
Leading strand Lagging strand
DNA Leading Strand Since DNA is always synthesized in the
5' to 3' direction and the template strands run antiparallel, only one strand is able to be built continuously, that is the Leading strand.
The 3'-5' proceeding leading strand uses a 5'-3‘ template because DNA Polymerase can "read" the template and continuously adds nucleotides
DNA Lagging Strand The other strand is synthesized
discontinuously in short fragments in the opposite direction to the replication fork and is known as the Lagging strand
The 5'-3‘ lagging strand uses a 3'-5‘ template because DNA Polymerase cannot "read" the template and continuously adds nucleotides.In the lagging strand the RNA Primase adds more RNA Primers.
Okazaki Fragments Short
fragments of DNA that are a result of the synthesis of the lagging strand during DNA replication
etc).
DNA Polymerase III The enzyme that adds free
deoxyribonucleotides from primer to primer forming Okazaki fragments
DNA Polymerase I The enzyme that removes RNA primers
from the leading and lagging strands and replaces the RNA primers with appropriate deoxyribonucleotides
DNA Ligase The enzyme that joins the
Okazaki fragments into one strand by creation of a phosphodiester bond
As the two strands of DNA are synthesized, two double-stranded DNA molecules are produced that automatically twist into a helix
DNA Replication Video