3.3 - DNA replication

Question 1

DNA replication

Answer 1

• synthesis of new DNA
• occurs during S-phase of the cell cycle
• semi-conservative
• 2 strands of parental double helix unwind, and each specifies a new daughter strand by base-pairing rules
• 3 Steps: Initiation, elongation (of the new strand), termination

Question 2

Initiation in DNA Replication

Answer 2

• Begins at an origin of replication (OriC)
• involves: initiator proteins, helicases, primase, polymerase III
• at the end of initiation: helicases are ready to unwind, RNA primers are in place, polymerases are ready for elongation

Question 3

Initiation Components: Initiator proteins

Answer 3

-separate the strands on OriC

Question 4

Initiation Components: Helicases

Answer 4

• loaded only

- 1 at each fork

Question 5

Initiation Components: Primase

Answer 5

-synthesizes 2 RNA Primers

Question 6

Initiation Components: Primer

Answer 6

• synthesized by primase
• short (4-6 nt) RNA
• complementary to DNA template
• antiparallel
• (attracts DNA polymerase to build)

Question 7

Initiation Components: Polymerase III

Answer 7

• loaded only

- 2 at each fork

Question 8

Elongation in DNA Replication

Answer 8

• Helicases unwind duplex bidirectionally
• DNA polymerase adds DNA nucleotides to the 3′ end
• involves: helicases, DNA polymerase III dimers, dNTPs, primases (RNA primers), DNA polymerae I, DNA Ligase

Question 9

Elongation Components: Helicases

Answer 9

• unwind duplex bidirectionally

- 2 forks move away from the origin

Question 10

Elongation Components: DNA polymerase III

Answer 10

• adds single nucleotides to 3’ OH of pre-existing polynucleotide basepaired to a DNA template
• 5’ > 3’ polymerase activity
• will also “proofread” 3’ - 5’

Question 11

Elongation Components: dNTPs

Answer 11

deoxynucleoside triphosphate precursors

• dATP – deoxyadenosine triphosphate
• dGTP – deoxyguanosine triphosphate
• dCTP – deoxycytidine triphosphate
• dTTP – deoxythymidine triphosphate

Question 12

Lagging strand

Answer 12

• lagging strand synthesis is discontinuous (okazaki fragments)
• Parent template is 5’ > 3’
• Contain primase that synthesizes RNA primer

Question 13

Elongation Components: DNA polymerase I

Answer 13

• Primer removal and gap filling in the completion of an Okazaki fragment
• at 3’OH of one Okazaki fragment (so can only go 5’ to 3’ direction)
  1) breaks phosphodiester “forward” (5’ > 3’)
  2) adds deoxynucleotide to 3’OH (5’ > 3’)
• these two activities alternate until primer is removed
• will also “proofread” 3’ - 5’

Question 14

Elongation Components: DNA Ligase

Answer 14

• nicks still exist between okazaki fragment, therefore…..
• forms the final phosphodiester linkage on lagging strand between 3’OH of one okazaki fragment and 5’PO4 of next okazaki fragment

Question 15

Mismatched base-pairs (proof-reading)

Answer 15

• mismatched bps create distortion in diameter detected by trailing portion of polymerase
• polymerizing activity stops
• 3’ to 5’ exonuclease activity breaks 3-4 phospodiester linkages including mismatched nucleotides
• resumes 5’ to 3’ polymerase activity

Question 16

Termination in DNA Replication

Answer 16

• stops DNA replication by addition of telomere sequence at end
• includes in circular chromosome: topoisomerase II
• includes in linear chromosome: telomerase, DNA polymerase I, DNA ligase

Question 17

Telomere Synthesis

Answer 17

-does not occur after chromosome replication in all cells

1) after telomerase extension of 3’ end
2) primase-primed DNA polymerase synthesis of other strand
3) ligase forms final phosphodiester linkage
4) repair polymerase removes primer

Question 18

Leading strand parent template

Answer 18

• parent runs runs 3’ > 5’

- therefore, leading strand synthesizes 5’ > 3’ w/o problems