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

DNA Replication Video