Chapter 12 - DNA Replication

Question 1

What are the three models of DNA replication and which is the acutal model?

Answer 1

Semiconservative replication - each strand of DNA serves as the template for synthesis for the new strand
- All DNA replication takes place in this manner

Conservative replication - the entire double strand of DNA serves as a template for new strand
- Original strand is fully conserved
- A model once considered before semiconservative

Dispersive replication - both nucleotide strands break down into fragments that serve as small templates for synthesis, and those fragment are then recombined after synthesis
- A model once considered before semiconservative

Question 2

What was the Meselson-Stahl experiment (7) and what did it discover?

Answer 2

Discovered the semiconservative nature of DNA replication

Experiment:
1. Added radiolabeled nitrogen so it could combine with nitrogenous bases in E. coli
2. Second round of replication in regular nitrogen medium
3. Additional round in regular nitrogen medium
4. Spun cultures via equilibrium density centrifugation
5. Found original E. coli grown in radiolabeled nitrogen medium had a single heavy band
6. Bacteria grown after one round of replication in regular nitrogen medium had an intermediate band because it contained one strand of radiolabeled nitrogen and one of regular nitrogen
7. As they did this with subsequent rounds of replication, they found a lighter band form, which became more intense with every replication because there is more regular nitrogen after each replication

Question 3

What is a replicon?

Answer 3

Segment of DNA undergoing replication

Each contains origin of replication

Question 4

What is theta replication and how does it work (5)?

Answer 4

Circular DNA replication in bacteria that results in two circular DNA molecules

Steps:
1. Double stranded DNA begins to unwind at origin of replication
2. Produces two single stranded template strands
3. A replication bubble forms and gets progressively larger because of unwinding
4. Replication fork proceeds around the circle
5. DNA is synthesized, resulting in two circular DNA molecules

Question 5

What is a replication fork?

Answer 5

The location where replication bubble becomes large enough and template strands have unwound enough

Question 6

What is linear replication and how does it work (3)?

Answer 6

Eukaryotic linear DNA replication that results in two linear DNA molecules

Steps:
1. Multiple origins of replication where DNA unwinds, producing a replication bubble
2. DNA synthesis takes place on both strands at each end of the bubble as the replication forks proceed outward
3. Eventually, the forks of adjacent bubbles run into each other and the segments of DNA fuse, producing two identical DNA molecules

Question 7

What are the 3 general requirements for DNA replication?

Answer 7

Template consisting of single-stranded DNA

Raw materials to be assembled into new strand (DNTPs)

Enzymes and proteins that read template and assemble materials into new molecule

Question 8

What is the direction of DNA replication?

Answer 8

Always occurs in 5’ to 3’ direction

Because strands are antiparallel, DNA replication has to occur in two directions

One goes with unwinding and one goes against

Question 9

What is the leading strand?

Answer 9

Strand that follows the direction of unwinding

As DNA unwinds, it exposes 3’ to 5’ strand in the direction of unwinding, so it can synthesize in 5’ to 3’ direction

Allows for continuous replication

Question 10

What is the lagging strand?

Answer 10

Strand that goes in direction opposite of unwinding

Template strand runs 5’ to 3’ in the direction of unwinding, so DNA has to be synthesized in the direction away from unwinding

Cannot have continuous repliation - has fragmented replication instead

Question 11

How is the lagging strand synthesized?

Answer 11

DNA synthesis starts at the fork, each time proceeding away from the fork

Once more strand is exposed, it moves back to the fork and replicates until it meets up with previous fragment

Fragments are called Okazaki fragments and are rejoined after replication

Question 12

How many origins of replication are there in bacterial DNA replication?

Answer 12

One origin of replication in a single circular chromosome - caled the oriC

Question 13

What are the four steps of DNA replication in bacteria?

Answer 13

Initiation, unwinding, elongation, and termination

Question 14

What occurs in initiation of DNA replication in bacteria?

Answer 14

Initiator proteins bind to oriC, causing a short stretch of DNA to unwind

Once some DNA is unwound, helicases come in and bind onto single stranded DNA

Question 15

What occurs during unwinding of DNA replication in bacteria?

Answer 15

DNA helicase binds to the lagging strand template at each replication fork and moves in the 5’ to 3’ direction along this strand, breaking hydrogen bonds and moving along the replication fork

Single-stranded-binding proteins stabilize the exposed single-stranded DNA
- Prevent structures like hairpins from forming

DNA gyrase (a topoisomerase) relieves strain ahead of the replication fork
- Creates less torque/supercoiling in the strand by making small cuts in the DNA

Question 16

What occurs during elongation of DNA replication in bacteria?

Answer 16

Each of the unwound single strands are used as a template

Primase synthesizes short primers, which are 10-12 nucleotides strands of RNA
- Provide DNA polymerase the 3’ OH groups that are needed to add DNA nucleotides
- Will be removed and replaced with DNA nucleotides later

On leading strand, only need one primer because replication is continuous

On lagging strand, need multiple primers - one is put at the start of each Okazaki fragment

After primer is placed, DNA polymerase can elongate strand
- After DNA polymerase II has added nucleotides, DNA polymerase I follows behind and replaces the RNA nucleotides of the primer with DNA nucleotides
- DNA polymerase I attaches to last nucleotide of preceding Okazaki fragment and replaces primer in 5’ to 3’ direction

Once DNA polymerase I replaces primer, there is a nick remaining between fragment
- DNA ligase seals nick with a new phosphodiester bond between phosphate group and OH group of the the two ends of adjacent fragments

Question 17

What is DNA polymerase III and its functions?

Answer 17

Large multiprotein complex that synthesizes DNA by adding nucleotides to 3’ end of newly synthesized/growing strand

Enzymatic activities:
- In 5’ to 3’ direction - adds new nucleotides
- In 3’ to 5’ direction - has proofreading capabilities in the form of exonuclease activity, which allows it to remove nucleotides - If it accidentally puts in wrong nucleotide, it can remove wrong one and put correct one in

High progressivity - moves quickly along strand for synthesis and allows for long strands of DNA to be synthesized

Question 18

What is the function of DNA polymerase I?

Answer 18

5’ to 3’ - adds nucleotides

Also 5’ to 3’ and 3’ to 5’ - exonuclease activity (removes nucleotides)
- Removes RNA primers and replaces them with DNA
- Makes it more accurate and efficient

Question 19

What are the two options for termination of DNA replication in bacteria?

Answer 19

Replication forks meet, terminating replication

Ter site (termination site) blocks replication from continuing

Question 20

What is the accuracy of DNA replication in bacteria?

Answer 20

End up with about 1 in 1 billion base pairs replicated incorrectly

Question 21

What are the 2 methods for accuracy in DNA replication in bacteria?

Answer 21

Polymerases are very particular in how they pair nucleotides with complements on template strand

DNA polymerase III has proofreading activity
- If it inserts the wrong base, it stalls replication from occurring, backs up, uses exonuclease activity to remove bad nucleotide, and replaces it with correct one

Accuracy goes from 1 in 100,000 to 1 in 10 million

Question 22

What are the 5 added challenges of DNA replication in eukaryotes?

Answer 22

Much larger genome to replicate

Multiple initiation sites

Linear chromosomes

Nucleosome assemblies as an obstacle

Replication at the telomeres

Question 23

What are the origins of replication in DNA replication in eukaryotes?

Answer 23

Vary greatly and primarily defined by modifications in chromatin structure

Origin recognition complex (ORC) binds to specific origins of replication to initiate replication
- Recruit helicase (in G1 phase) to strand to separate double stranded DNA (in S phase) - starts to unwind DNA after G1/S checkpoint is passed
- Replication managed/coordinated with cell cycle

Question 24

What are the 5 stages of DNA replication in eukaryotes?

Answer 24

Initiation

Unwinding

Strand synthesis with DNA polymerases

Nucleosome assembly

Replication of telomeres

Question 25

What occurs during intiation of DNA replication in eukaryotes?

Answer 25

Licensing (G1)
- Origins of replication being recognized by ORCs are approved for replication
- Replication licensing factors bind to each origin along the DNA
- When ORCs bind and licensing factors bind, two other licensing factors bind

Replication intiates (S phase)
- All bound machinery initiate replication at the origins of replication

Question 26

What occurs during unwinding of DNA replication in eukaryotes?

Answer 26

Topoisomerases (DNA gyrase) relieve torque

Helicase unwinds strand - bidirectional

Question 27

What occurs during strand synthesis with DNA polymerases in DNA replication in eukaryotes?

Answer 27

Polα - initiates DNA synthesis by laying down RNA primer and synthesizing a few DNA nucleotides after primer

Polδ - replicates leading strand

Polε - replicates lagging strand

Translesion polymerases - have lower fidelity and accuracy and bypass errors and distortions

Question 28

What occurs during nucleosome assembly of DNA replication in eukaryotes?

Answer 28

Chromatin structure is disrupted by replication fork as it goes through DNA

Nucleosomes are quickly disassembled and reassembled on new DNA

Original histone octamers are reassembled into two dimers and a tetramer

Histone chaperones (ex: CAF1) shuttle histone dimers and tetramers randomly to one of the newly synthesized strands

Other dimers and tetramers floating around the cell are brought in and moved by chaperones in order to reform octamers

Question 29

How can replication of telomeres occur in DNA replication in eukaryotes and in which types of cells does it occur?

Answer 29

Telomeres have repeated sequences on 3’ end called the G-rich overhang

Can be extended using telomerase
- Have protein and RNA components
- RNA components have complementary sequneces that allow it to beind to strand, allowing nucleotides to be added and the strand to be elongated
- Telomerase is reoved and telomere length is retained (not shortened)

Somatic cells lack telomerase, so they will die after a set amount of replications

Germ cells have telomerase, so they will be able to continually replicate

Question 30

What is the end-replication problem in DNA replication of eukaryotes?

Answer 30

Initial RNA primers at ends of chromosomes are removed but never replaced - end up with non-synthesized DNA at the end of chromosomes

Every time replication occurs, the telomeres shrink because they are not being replicated