Lecture 2: DNA REPLICATION IN EUKARYOTES AND PROKARYOTES Flashcards
All the Genetic Information Must Be Accurately
Copied Every Time a Cell Divides:
What are the 4 properties of DNA replication?
1 * Rapid
2 * Accurate
3 * Highly regulated in eukaryotes
4 * Semiconservative
What is summary of DNA replication?
(DNA helix unwinds and separates into 2 single strands which form the
templates for the new strands to be synthesised, complementary to the existing ones.
Stages of the cell cycle?
Original cell
m = Mitosis
2 daughter cells
GAP - 1
S =DNA synthesis
GAP - 2
M
What are REPLICONS?
- A segment of DNA that
undergoes replication is called a replicon
2 * Each replicon contains an origin of replication
3 * Different organisms use different modes of
replication
Different organisms use different modes of
replication: includes
- Theta replication: the two nucleotide strands of a circular DNA molecule unwind at the origin, creating a replication bubble (circular DNA – bacteria)
- Rolling circle replication: initiated by a break in one strand of circular DNA, which produces a 3 ′ -OH group to which new nucleotides are added (some viruses & F factor)
- Linear eukaryotic replication: Linear eukaryotic DNA contains many origins of replication. Unwinding and replication take place on both templates at both ends of the replication bubble until adjacent replicons meet
Different organisms use different modes of
replication:
EXPLAIN Theta Replication:
the two nucleotide strands of a circular DNA molecule unwind at the origin, creating a replication bubble (circular DNA – bacteria)
Leading strand
1. DNA UNWINDS AT THE ORIGIN
Lagging strand
Unwinding and replication
2. AT EACH FORK, DNA SYNTHESIS OF THE LEADING STRAND PROCEEDS CONTINUOUSLY IN THE SAME DIRECTION AT THAT OF UNWINDING
Leading strand
3. DNA SYNTHESIS OF THE LAGGING STRAND PROCEEDS DISCONTINUOUSLY IN THE DIRECTION OPPOSITE THAT OF UNWINDING
Different organisms use different modes of
replication:
EXPLAIN Rolling circle replication:
initiated by a break in one strand of circular DNA, which produces a 3 ′ -OH group to which new
nucleotides are added (some viruses & F factor)
Leading strand
1. CONTINUOUS DNA SYNTHESIS BEGINS AT THE 3’ END OF THE BROKEN NUCLEOTIDE STRAND
- AS THE DNA MOLECULE UNWINDS, THE 5’ END IS PROGRESSIVELY DISPLACED
unwinding and replication
Different organisms use different modes of
replication:
EXPLAIN Linear eukaryotic replication:
Linear eukaryotic DNA contains
many origins of replication. Unwinding and replication take place on both templates at both ends of the replication bubble until adjacent replicons meet
- AT EACH FORK, THE LEADING STRAND IS SYNTHESISED CONTINUOUSLY IN THE SAME DIRECTION AS THAT OF UNWINDING
leading strand
lagging strand - THE LAGGING STRAND IS SYNTHESISED DISCONTINUOUSLY IN THE DIRECTION OPPOSITE THAT OF UNWINDING
The large linear chromosomes in eukaryotic cells contain far too much
DNA to be replicated speedily from a single origin
The large linear chromosomes in eukaryotic cells contain far too much
DNA to be replicated speedily from a single origin
What does DNA SYNTHESIS REQUIRE? 4
- DNA TEMPLATE
- new DNA is synthesised from deoxyribonuclease triphosphate (dNTPs) - DEOXYRIBONUCLEOSIDE TRIPHOSPATES
- in replication, the 3’-OH group of the last nucleotide on the strand attacks the 5’-phosphate group of the incoming dNTP. - A GROWING NUCLEOTIDE STRAND
- 2 phosphates are cleaved off - ENZYMES AND PROTEINS
- a phosphodiester bond forms between the two nucleotides.
SUMMARY:New DNA is synthesized from deoxyribonucleoside triphosphates (dNTPs). The newly synthesized strand is complementary and antiparallel to the template strand. The two strands are held together by hydrogen bonds (represented by red dotted lines) between the bases
Replication takes place in four stages
WHAT ARE THEY?
WHAT ELSE DO THEY REQUIRE?
- Initiation
- Unwinding - HELICASE
- Elongation
- Termination
And requires a large number of enzymes and proteins
INITIATION STEPS:
: Replication
begins when an initiator
protein binds to an origin of
replication
- initiator proteins (DnaA) bind to oriC, the origin of replication…
- …causing a short of DNA to unwind
- the unwinding allows helicase and other single-strand-binding proteins to attach to the single-stranded DNA
UNWINDING STEPS
Helicase unwinds the DNA.
- DNA helicase binds to the lagging-strand template a each replication fork and moves in the 5’ TO 3’ direction along this strand, breaking hydrogen bonds and moving the replication fork.
- Single-strand-binding proteins stabilise the exposed single-stranded DNA.
- DNA GYRASE relives strain ahead of the replication fork
Elongation steps
Elongation: Primase synthesises short primers consisting of RNA
nucleotides, providing a 3 ′ -OH group for DNA polymerase
Elongation: DNA polymerase I removes and replaces primers; DNA ligase seals the nicks
- Primase synthesises short stretches of the RNA nucleotides, providing a 3’OH group to which DNA polymerase can add DNA nucleotides.
DNA SYNTHESIS
- On the leading strand, where replication is continuous, a primer is required only at the 5’ end of the newly synthesised strand.
DNA SYNTHESIS CONTINUES
- on the lagging strand, where replication is discontinuous, a new primer must be generated at the beginning of each Okazaki fragment.
Elongation: DNA polymerase I removes and replaces primers;
DNA ligase seals the nicks
Template strand
- DNA nucleotides have been added to the primer by DNA polymerase III
- DNA polymerase I replaces the RNA nucleotides of the RNA primer has been replaced, a nick remains in the sugar-phosphate backbone of the strand.
- DNA ligase seals this nick with a phophosdiester bond between the 5’ phosphates group of the initial nucleotides added by DNA polymerase II and the 3’OH group of the final nucleotides added by DNA polymerase I.
The major enzymatic components of elongation interact at the replication fork
The major enzymatic components of elongation interact at the replication fork
Initiator protein
Binds to origin and separates strands of DNA to initiate replication
DNA helicase
unwinds DNA at replication fork
Single-strand-binding proteins
attach to single-stranded DNA and prevent secondary structures from forming
DNA gyrase
Moves ahead of the replication fork, making and resealing break in the double-helical DNA to release the torque that builds up as a result of unwinding at the replication fork.
DNA primase
Synthesizes a short RNA primer to provide a 3’-OH group for the attachment of DNA nucleotides
DNA Polymerase III
Elongates a new nucleotide strand from the 3’-OH group provided by the primer
DNA Polymerase I
Removes RNA primers and replaces them with DNA
DNA ligase
Joins Okazaki fragments by sealing breaks in the sugar-phosphate backbone to newly synthesised DNA
Understanding Termination of replication; 2
- In some DNA molecules, replication is terminated whenever two
replication forks meet. - In others, specific termination sequences (called Ter sites) block
further replication.
Eukaryotic replication has some extra challenges; WHAT ARE THEY?
- 3
- Eukaryotic GENOMES much LARGER, so have MULTIPLE POINTS OF ORIGIN OF REPLICATION. Regulated through licensing of the origins.
- Eukaryotic DNA is ASSOCIATED WITH HISTONE PROTEINS proteins to form nucleosomes, which must be RE-ASSEMBLED AFTER REPLICATION of the DNA.
- Eukaryotic chromosomes are LINEAR, whereas prokaryotic chromosomes
are circular. The ends of linear chromosomes are replicated in some
cells by the enzyme telomerase, so that they DO NOT SHORTEN.
Eukaryotic cells contain more different DNA polymerases than bacteria do
Eukaryotic cells contain more different DNA polymerases than bacteria do
In eukaryotic cells, replication is coordinated with the cell cycle HOW? 3
- multiple points of origin of replication
- ALL the DNA needs to be copied & only once
- initiation of replication divided into two steps:
1. the origins are licensed — approved for
replication (G1).
2. the replication machinery initiates replication at
each licensed origin (S).
ORC
origin recognition complex
CDC6
Cell division cycle 6 (S-phase)
MCM
Mitochromosome maintenance (hexamer of MCM 2–7) = Helicase
CDT1
CDC-dependent transcript 1 (S-phase)
Eukaryotic DNA is licensed at origins
EXPLAIN HOW?
- Assembly of the replisome is ordered & begins at precise chromosomal locations = origins.
2 * Origins: Poorly understood. AT-rich. Vary between species.
3 * ORC (origin recognition complex) binds the origin
4 * The ORC, with the help of two additional licensing factors,
allows a complex called MCM2-7 (for minichromosome
maintenance) to bind to an origin
5 * In S phase, MCM2-7 complex associates with cell-phase Specific cofactors and forms an active helicase that
unwinds double-stranded DNA for replication
In eukaryotes, nucleosomes must be assembled
after DNA replication…HOW
CAF-1 (chromatin assembly factor 1) brings histones to the replication fork - assemble to form nucleosomes
PCNA = proliferating cell nuclear antigen = clamp
Linear chromosomes have ends SUMMARY
Linear chromosomes
have an ‘end replication
problem’ – they can’t
replace the RNA primers
with DNA nucleotides at
their ends because
there is no free 3’-OH
group
Linear chromosomes have ends
A. Circular DNA
Replication around the circle provides a 3’-OH group in front of the primer; nucleotides can be added to the 3’-OH group when the primer is replaced.
B. Linear DNA
1. In linear DNA with multiple origins of replication, elongation of DNA in adjacent replicons provides a 3; -OH group for replacement of each primer.
- The terminal primer is positioned 70-100 nucleotides from the end of the chromosome…
- …Leaving a gap that is not replicated.
The ENZYME TELOMERASE is
responsible for the replication of
chromosome ends.
- the telomere has a protruding end with G-rich repeated sequence.
- The RNA part of telomerase is complementary to the G-rich strand and pairs with it, providing a template for the synthesis of copies of the repeats.
- After several nucleotides have been added, the RNA template moves along the DNA.
- More nucleotides are added.
- The telomerase is removed.
- Synthesis takes place on the complementary strand, filling in the gap due to the removal of the RNA primer at the end.
Telomeres play a role in aging EXPLAIN
1 * In genetically engineered mice lacking a functional telomerase
gene - progressive telomere shortening in successive
generations. After several generations, these mice show signs
of premature aging.
2 * In somatic cells that express telomerase, telomeres do not
shorten, cell aging is inhibited, and the cells divide indefinitely.
3 * Some diseases are associated with abnormalities of telomere
replication. Eg Werner syndrome:
Werner syndrome:
- Signs of premature aging in adolescence.
2 * People develop cancer, osteoporosis, heart and artery disease.
3 * The causative gene, WRN, encodes a RecQ helicase enzyme, which is
necessary for the efficient replication of telomeres.
Fidelity of DNA replication: mistakes are made – but
not very often
WHAT ARE THE Several mechanisms ensure the high rate of accuracy; 3
Several mechanisms ensure the high rate of accuracy
1 - precise nucleotide selection
2 - proofreading
3 - mismatch repair
