Week 7 (DNA Replication)

Question 1

DNA structure

Answer 1

-AG(purines)CT(pyrimidines), AT and GC
-3 HB pair GC, 2 HB pair AT
-anti-paralel 5’-3’
-right-handed double helix

Question 2

How is DNA semi-conservative

Answer 2

after one round of DNA replication, each DNA has 1 parental and 1 newly synthesized strand

Question 3

3 modes of DNA replication

Answer 3

Rolling Circle: specialized form that occurs in the F factor of E Coli + some viruses (1 origin of replication)

Theta: occurs in most circular DNA

Linear: in linear chromosomes of eukaryotic cells

Question 4

Rolling circle replicaiton

Answer 4

1. initiated by break in 1 nucleotide strand
2. DNA synthesis begins at 3’ end of broken strand (inner template strand), 5’ end displaced
3. cleavage releases single-stranded linear DNA + a souble stranded circular DNA
4. Linear DNA may circularize + serve as a template for synthesis of complimentary strand

Products: multiple circular DNA molecules
Uni-directional

Question 5

Theta replication

Answer 5

1. double-stranded DNA unwinds at rep origin
2. produces single-stranded templates for synthesis of new DNA, replication bubble forms
3. forks proceed around circle

Products; 2 circular DNA molecules
Uni or bi directional

Question 6

Linear replication

Answer 6

1. each chromosome contains numerous origins
2. at each DNA unwinds, produces replication bubble
3. DNA synthesis occurs on both strands at each end of bubble, as replication fork proceeds outwards

Products: 2 identical linear DNA molecules
Bidirectional

Question 7

What are the features that DNA replication require?

Answer 7

-DNA polymerase
-4 deoxyribonucleoside triphosphate (dNTPs)
-single-stranded template of DNA
-RNA primer (provides 3’-OH end)

Question 8

What are the proteins involved in prokaryotic DNA replication?

Answer 8

-DnaA: initiators that bind to oriC (origin in E. coli) that causes a short stretch of DNA to unwind which allows helicase and other SSB’s to attach to single-stranded DNA
-DNA helicase: unwinds 5’-3’ coated with SSBs (travels only on lagging strand)
-DNA gyrase: topoisomerase that relieves tension from helicase
-primase: binds to helicase 1, synthesized short RNA primer
-DNA Pol III: main polymerase, has 5’-3’ polymerase activity and 3’-5’ exonuclease activity (can add dNTP to 3’ end and remove misplaced nucleotides)
-DNA Pol I: similiar to Pol III but also has 5’-3’ exonucleuse activity and can replace RNA primer

Question 9

Describe the Lagging and Leading strands

Answer 9

Lagging:
When DNA synthesis by Pol. III reaches the 5’ end of
the RNA primer, Pol. III is “swapped” for Pol. I.
* DNA Polymerase I (with its 5’→ 3’ exonuclease
activity) removes the RNA primer and re-synthesizes
a short tract of DNA.
* DNA ligase makes a phosphodiester bond between
the 5’ phosphate and the 3’ OH group.
* Okazaki fragments are thus joined together to make
one continuous length of DNA
5. Proteins involved in Prokaryotic DNA replication

Leading: DNA synthesis continuous by DNA Poly III

Question 10

Prokaryotic DNA replication steps

Answer 10

1. DnaA initiator proteins bind to oriC and opens up the DNA
2. DNA helicase binds lagging strand to unwind DNA
3. Single stranded DNA binding proteins bind to keep DNA single stranded
4. DNA gyrase binds upstream of the unwinding fork to prevent torsion
5. • Primase (RNA polymerase) synthesizes a short RNA primer that provides the 3’ OH end for DNA polymerase to begin DNA synthesis (Primase binds to
     the helicase)
6. DNA Poly III is continuously building onto leading strand, lagging occurs in sections
7. DNA Poly I replaces primers with DNA
8. DNA ligase connects fragments by sealing breaks

Question 11

Eukaryotic cell cycle

Answer 11

• Eukaryotic origins are “prepared” for
  replication in the G1 phase (called “origin
  licensing”).
• Replication licensing factors attach to each
  origin of replication during G1 phase
• Replication begins in the S phase.
• Allows replication of entire genome once
  and only once.

Question 12

Eukaryotic chromosome replication

Answer 12

Replication begins at autonomously replicating sequences
* Bidirectional replication from multiple origins of replication on each chromosome
* Helicase binds to initiator protein on double stranded DNA, involves the MCM complex of proteins
* Shorter RNA primers and shorter Okazaki fragments
* DNA replication only occurs during S phase
* Multiple DNA Polymerases
* Pol α (alpha) has primase activity (generates the RNA primer)
* Pol ε (epsilon) performs leading strand replication
* Pol δ (delta) performs lagging strand replication
* Nucleosomes (two each of histones H2A, H2B, H3, H4 plus H1): need to be removed from parental DNA and properly re-assembled on newly-synthesized DNA
* Telomeres: shorten at each round of eukaryotic replication

Question 13

Telomerase

Answer 13

Telomerase activity extends
eukaryotic chromosome ends
in replicating cells

Telomerase can extend the 3’ end without
the use of a template.
* Gap remains, doesn’t matter since end of chromosome is extended at
each replication.
* Chromosome does not become shorter