Lecture 2: DNA REPLICATION IN EUKARYOTES AND PROKARYOTES

Question 1

All the Genetic Information Must Be Accurately
Copied Every Time a Cell Divides:

What are the 4 properties of DNA replication?

Answer 1

1 * Rapid
2 * Accurate
3 * Highly regulated in eukaryotes
4 * Semiconservative

Question 2

What is summary of DNA replication?

Answer 2

(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.

Question 3

Stages of the cell cycle?

Answer 3

Original cell
m = Mitosis
2 daughter cells

GAP - 1
S =DNA synthesis
GAP - 2
M

Question 4

What are REPLICONS?

Answer 4

1. 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

Question 5

Different organisms use different modes of
replication: includes

Answer 5

1. Theta replication: the two nucleotide strands of a circular DNA molecule unwind at the origin, creating a replication bubble (circular DNA – bacteria)
2. 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)
3. 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

Question 6

Different organisms use different modes of
replication:

EXPLAIN Theta Replication:

Answer 6

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

Question 7

Different organisms use different modes of
replication:

EXPLAIN Rolling circle replication:

Answer 7

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

2. AS THE DNA MOLECULE UNWINDS, THE 5’ END IS PROGRESSIVELY DISPLACED
   unwinding and replication

Question 8

Different organisms use different modes of
replication:

EXPLAIN Linear eukaryotic replication:

Answer 8

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

1. AT EACH FORK, THE LEADING STRAND IS SYNTHESISED CONTINUOUSLY IN THE SAME DIRECTION AS THAT OF UNWINDING
   leading strand
   lagging strand
2. THE LAGGING STRAND IS SYNTHESISED DISCONTINUOUSLY IN THE DIRECTION OPPOSITE THAT OF UNWINDING

Question 9

The large linear chromosomes in eukaryotic cells contain far too much
DNA to be replicated speedily from a single origin

Answer 9

The large linear chromosomes in eukaryotic cells contain far too much
DNA to be replicated speedily from a single origin

Question 10

What does DNA SYNTHESIS REQUIRE? 4

Answer 10

1. DNA TEMPLATE
   - new DNA is synthesised from deoxyribonuclease triphosphate (dNTPs)
2. DEOXYRIBONUCLEOSIDE TRIPHOSPATES
   - in replication, the 3’-OH group of the last nucleotide on the strand attacks the 5’-phosphate group of the incoming dNTP.
3. A GROWING NUCLEOTIDE STRAND
   - 2 phosphates are cleaved off
4. 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

Question 11

Replication takes place in four stages

WHAT ARE THEY?
WHAT ELSE DO THEY REQUIRE?

Answer 11

1. Initiation
2. Unwinding - HELICASE
3. Elongation
4. Termination

And requires a large number of enzymes and proteins

Question 12

INITIATION STEPS:

Answer 12

: Replication
begins when an initiator
protein binds to an origin of
replication

1. initiator proteins (DnaA) bind to oriC, the origin of replication…
2. …causing a short of DNA to unwind
3. the unwinding allows helicase and other single-strand-binding proteins to attach to the single-stranded DNA

Question 13

UNWINDING STEPS

Answer 13

Helicase unwinds the DNA.

1. 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.
2. Single-strand-binding proteins stabilise the exposed single-stranded DNA.
3. DNA GYRASE relives strain ahead of the replication fork

Question 14

Elongation steps

Answer 14

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

1. Primase synthesises short stretches of the RNA nucleotides, providing a 3’OH group to which DNA polymerase can add DNA nucleotides.

DNA SYNTHESIS

2. 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

3. on the lagging strand, where replication is discontinuous, a new primer must be generated at the beginning of each Okazaki fragment.

Question 15

Elongation: DNA polymerase I removes and replaces primers;
DNA ligase seals the nicks

Answer 15

Template strand
- DNA nucleotides have been added to the primer by DNA polymerase III

2. 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.
3. 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.

Question 16

The major enzymatic components of elongation interact at the replication fork

Answer 16

The major enzymatic components of elongation interact at the replication fork

Question 17

Initiator protein

Answer 17

Binds to origin and separates strands of DNA to initiate replication

Question 18

DNA helicase

Answer 18

unwinds DNA at replication fork

Question 19

Single-strand-binding proteins

Answer 19

attach to single-stranded DNA and prevent secondary structures from forming

Question 20

DNA gyrase

Answer 20

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.

Question 21

DNA primase

Answer 21

Synthesizes a short RNA primer to provide a 3’-OH group for the attachment of DNA nucleotides

Question 22

DNA Polymerase III

Answer 22

Elongates a new nucleotide strand from the 3’-OH group provided by the primer

Question 23

DNA Polymerase I

Answer 23

Removes RNA primers and replaces them with DNA

Question 24

DNA ligase

Answer 24

Joins Okazaki fragments by sealing breaks in the sugar-phosphate backbone to newly synthesised DNA

Question 25

Understanding Termination of replication; 2

Answer 25

1. In some DNA molecules, replication is terminated whenever two replication forks meet. 2. In others, specific termination sequences (called Ter sites) block further replication.

Question 26

Eukaryotic replication has some extra challenges; WHAT ARE THEY? - 3

Answer 26

1. Eukaryotic GENOMES much LARGER, so have MULTIPLE POINTS OF ORIGIN OF REPLICATION. Regulated through licensing of the origins. 2. Eukaryotic DNA is ASSOCIATED WITH HISTONE PROTEINS proteins to form nucleosomes, which must be RE-ASSEMBLED AFTER REPLICATION of the DNA. 3. 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.

Question 27

Eukaryotic cells contain more different DNA polymerases than bacteria do

Answer 27

Eukaryotic cells contain more different DNA polymerases than bacteria do

Question 28

In eukaryotic cells, replication is coordinated with the cell cycle HOW? 3

Answer 28

* 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).

Question 29

ORC

Answer 29

origin recognition complex

Question 30

CDC6

Answer 30

Cell division cycle 6 (S-phase)

Question 31

MCM

Answer 31

Mitochromosome maintenance (hexamer of MCM 2--7) = Helicase

Question 32

CDT1

Answer 32

CDC-dependent transcript 1 (S-phase)

Question 33

Eukaryotic DNA is licensed at origins EXPLAIN HOW?

Answer 33

1. 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

Question 34

In eukaryotes, nucleosomes must be assembled after DNA replication...HOW

Answer 34

CAF-1 (chromatin assembly factor 1) brings histones to the replication fork - assemble to form nucleosomes PCNA = proliferating cell nuclear antigen = clamp

Question 35

Linear chromosomes have ends SUMMARY

Answer 35

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

Question 36

Linear chromosomes have ends

Answer 36

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. 2. The terminal primer is positioned 70-100 nucleotides from the end of the chromosome... 3. ...Leaving a gap that is not replicated.

Question 37

The ENZYME TELOMERASE is responsible for the replication of chromosome ends.

Answer 37

1. the telomere has a protruding end with G-rich repeated sequence. 2. 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. 3. After several nucleotides have been added, the RNA template moves along the DNA. 4. More nucleotides are added. 5. The telomerase is removed. 6. Synthesis takes place on the complementary strand, filling in the gap due to the removal of the RNA primer at the end.

Question 38

Telomeres play a role in aging EXPLAIN

Answer 38

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:

Question 38

Werner syndrome:

Answer 38

1. 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.

Question 39

Fidelity of DNA replication: mistakes are made – but not very often WHAT ARE THE Several mechanisms ensure the high rate of accuracy; 3

Answer 39

Several mechanisms ensure the high rate of accuracy 1 - precise nucleotide selection 2 - proofreading 3 - mismatch repair