Download DNA Replication

DNA REPLICATION
Understandings:
• Nucleosomes help to supercoil the DNA.
• DNA structure suggested a mechanism for DNA replication.
• DNA polymerases can only add nucleotides to the 3’ end of a primer.
• DNA replications is continuous on the leading strand and discontinuous on the
lagging strand.
• DNA replication is carried out by a complex system of enzymes.
• Some regions of DNA do not code for proteins but have other important
functions.
Applications and Skills
• Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication in
preparation of samples for base sequencing.
• Tandem repeats are used in DNA profiling.
• Analysis of results of the Hershey and Chase experiment providing evidence that
DNA is the genetic material
Semi-Conservative Replication of DNA
■ The replication of DNA is semi-conservative and depends on
complementary base pairing.
■ The two strands of DNA separate (by breaking the hydrogen
bonds).
■ These two strands will serve as a template to create a
complementary strand.
■ The newly synthesized strand will contain an original strand
and new copy.
Alternative Theories:
■ Conservative replication – both strands of the parent DNA
remain together and another molecule is produced.
■ Dispersive replication – Every molecule produced by DNA
replication has a mixture of old and new sections in both of
its strands.
Meselson and Stahl’s Experiments
■ They cultured E.coli bacteria for many generations in a
medium where only nitrogen source (15N), so the nitrogen
bases of the bacterial DNA was 15N.
■ Transferred to a less dense 14N medium.
■ Spun for 24 hours in a centrifuge.
■ DNA showed up as a dark band in UV light.
Enzymes involved in DNA replication
Gyrase
■ Relieves strain created by the unwinding of DNA by helicase.
Helicase
■ Unwinds and separates the double-stranded DNA (creates a
replication fork).
Single Stranded Binding Proteins
■ Prevents separated strand from re-annealing.
DNA Primase
■ Generates a short RNA sequence (primer) to initiate DNA
synthesis.
DNA Polymerase III
■ Extends new strands in a 5’  3’ direction by joining
nucleotides together.
Okazaki Fragments
■ The lagging strand is copied away from the replication fork
in short fragments.
DNA Polymerase I
■ Removes RNA primers on lagging strand and replaces with
DNA nucleotides
DNA Ligase
■ Joins Okazai fragments together with phosphodiester
bonds.
■ Stage 1: DNA gyrase moves in before helicase and relives strains in the DNA
molecule.
■ Stage 2: Helicase uncoils the DNA double helix and splits it into two template
strands.
■ Stage 3: DNA polymerase III adds nucleotides in a 5’ to 3’ direction. (On the leading
strand it moves in the same direction as the replication fork)
■ Stage 4: DNA primase adds a short length of RNA attached by base pairing to the
template strand of DNA.
■ Stage 5: DNA polymerase III starts replication next to the RNA primer and adds
nucleotides in a 5’ to 3’ direction. (on the lagging strand, it moves away from the
replication fork).
■ Stage 6: Short lengths of DNA are formed between RNA primers on the lagging
strand, called Okazaki fragments.
■ Stage 7: DNA polymerase I removes the RNA primer and replaces it with DNA. A nick
is left in the sugar-phosphate backbone of the molecule where two nucleotides are
still unconnected.
■ Stage 8: DNA ligase seals up the nick by making another sugar-phosphate bond.
Stages in DNA replication
The leading and lagging strand
■ Because the two strands of the DNA double helix are
arranged in an anti-parallel fashion, synthesis on the two
strands occurs in very different ways.
■ Leading strand: is made continuously following the fork as it
opens.
■ Lagging strand: is made in fragments moving away from the
replication fork.
– New fragments are created on the lagging strand as the
replication fork exposes more of the template strand.
– These fragments are called Okazaki fragments.
Direction of Replication
■ DNA replication begins at sites called origins of replication.
■ In prokaryotes there is one site, in Eukaryotes there is
multiple.
■ Replication occurs in both directions away from the origin.
■ The results appear as a replication bubble.
■ Free nucleotides (deoxynucleoside triphosphate) are added
to the 3’ end.
Non-coding regions
■ Majority of the human genome is comprised of non-coding DNA
■ Only 1.5% of our genes code for proteins.
■ Examples:
– Telomeres – Regions of repetitive DNA at the end of a chromosomes,
protects against chromosomal deterioration
– Introns – Non-coding sequences within genes, are removed by RNA
splicing prior to the formation of mRNA.
– ncRNA genes – codes for RNA molecules that are not translated into
proteins, example: genes for tRNA
– Gene regulatory sequences (gene expression) – sequences that are
involved in the process of transcription, includes promoters, enhancers
and silencers
What if we want to stop replication….
DNA Sequencing
■ Nucleotides containing dideoxyribonucleic acid to stop DNA
replication in preparation for base sequencing.
■ DNA sequencing refers to the process by which the base
order of a nucleotide sequence is elucidated.
■ The most widely used for DNA sequencing involves the use
of chain-terminating dideoxynucleotides.
Dideoxynucleotides
■ Dideoxynucleotides (ddNTPs) lack the 3’-hydroxyl group
necessary for forming a phosphodiester bond.
■ ddNTPs prevent further elongation of a nucleotide chain
and effectively terminate replication.
■ The resulting length of a DNA sequence will reflect the
specific nucleotide position at which the ddNTP was
incorporated.
Sequencing
Tandem repeats
■ A variable number of tandem repeat is a short nucleotide
sequence that shows variation between individuals in terms
of the number of times the sequence is repeated.
■ Can be inherited as an allele.
■ Used in DNA profiling.
Nucleosomes
■ Nucleosomes help to supercoil DNA.
■ Eukaryotic DNA is associated with proteins called histones.
■ Histones help package the DNA into structures called
nucleosome.
■ Nucleosome consist of a central core of eight histone
protein with DNA coiled around the proteins.
■ Supercoiling allows a great length of DNA to be packed into
a much smaller space within the nucleus.