DNA Replication

Question 1

What type of replication is DNA replication?

Answer 1

Semi-conservative:

2 parent -> 1parent/1new + 1parent/1new -> 1parent/1new + 2new + 2new + 1parent/1new

Question 2

How was semi-conservative DNA replication found?

Answer 2

• Matthew Meselson and Franklin Stahl, 1958 experiment
• Grew bacteria with 15N media, then switched to 14N media (any new bacteria made has to use 14N)
• Looked at the distribution of 14N and 15N in the DNA molecules after multiple replications (each subsequent generation became more and more 14N with less and less 15N, but 15N still present)

Question 3

How is DNA replicated?

Answer 3

DNA polymerase catalyzes addition of deoxyribonucleotides to a DNA strand

Question 4

DNA Polymerase I (of E. coli)

Answer 4

Function: Primer removal and DNA repair

Additional enzyme activities: 5’ -> 3’ exonuclease

Question 5

DNA Polymerase III (of E. coli)

Answer 5

Function: Replication, catalyzes the strand-elongation reaction, uses hydrolysis of PPi + H2O -> 2Pi to make even more favourable, uses induced fit (conformational change)
Addition enzyme activities: 3’ -> 5’ exonucleoase

Question 6

What is the function of MG2+ in strand-elongation reaction?

Answer 6

Helps incorporate incoming nucleotides

- Required for catalysis!

Question 7

DNA Polymerase Requirements/Facts??

Answer 7

1) Requires dNTPs and Mg2+
2) New DNA strand is assembled on an existing template
3) Requires a primer to begin synthesis
4) Elongation is in 5’ to 3’ direction
5) Can correct mistakes using 3’ to 5’ exonuclease activity (proofreading)

Question 8

How are mistakes corrected by polymerase?

Answer 8

• DNA Pol I 3’-5’ exonuclease removes incorrect nucleotides from 3’ end by hydrolysis
• A mismatch results in a “stall” = pause gives enough time for incorrect region to flop into exonuclease active site where it is removed

Question 9

How is DNA separated into single strands?

Answer 9

Helicase: ring-like hexamers that use ATP to unwind DNA

Question 10

Building of the pre-priming complex

Answer 10

1) DnaA binds to the origin of replication locus (AT-rich areas, with consensus sequence)
2) DnaAs oligomerize (wrapping origin around themselves)
3) DnaB joins DnaA
4) DnaB is a helicase -> uses ATP hydrolysis to unwind the duplex, including AT-rich regions
5) Single-strand-binding proteins (SSB) bind to newly generated single-strand regions (prevents reformation of the double helix)
Result: pre-priming complex (makes single-stranded DNA accessible to other enzymes for the beginning of the synthesis of complementary strands)

Question 11

Priming

Answer 11

RNA polymerase called primase makes an RNA primer

Question 12

Synthesis of the leading and lagging strand

Answer 12

At the replication fork
Leading strand: continuous
Okazaki fragments: discontinuous
Due to antiparallel, always synthesized 5’ to 3’

Question 13

DNA polymerase III holoenzyme structure:

Answer 13

Clamp loader (in centre under DnaB): feeds DNA through and keeps the enzyme attached to DNA
DnaB (just on top of clamp loader): helicase 
2 Polymerase cores (on either side of clamp loader): contain B2 and exonuclease regions
Sliding clamps (B2): help polymerase "stay on task"

Question 14

Leading strand in trombone model

Answer 14

Made continuously by Pol III in 5’-3’ direction

Question 15

Lagging strand in trombone model:

Answer 15

• Is looped out
• Pol III adds ~1000 nucleotides in 5’-3’ direction
• Releases sliding clamp
• New loop formed, sliding clamp added
• Primase adds RNA primer
• Pol III makes new Okazaki fragment
• Pol I fills gaps between fragments and removes RNA primer with 5’-3’ exonuclease activity
• Ligase seals fragments

Question 16

Ligase Seals Okazaki Fragments by:

Answer 16

Joining the 3’OH group and the 5’phosphate group of two fragments
- Hydrolyzes ATP