DNA replication and repair

Question 1

What are the enzymes involved in prokaryotic DNA replication?

Answer 1

DnaA, DnaB, SSB, primosome

Question 2

What is the function of DnaA?

Answer 2

DnaA - recognizes and binds OriC – begins unwinding

- recruits other proteins

Question 3

What is the function of DnaB?

Answer 3

DnaB – major helicase – ring shaped hexamer

ATP-dependent motor that unwinds DNA

Question 4

What is the function of SSB?

Answer 4

SSB – single stranded DNA binding protein – protects exposed ssDNA

Question 5

What is the function of the primosome?

Answer 5

Primosome – multi-protein complex that contains DnaG
(a primase) – makes short RNA fragments complementary
to the DNA – these are primers for replication

Question 6

What is the structure of prokaryotic DNA Pol III?

Answer 6

3 important pieces

• Core – includes α subunit – this is the polymerase
• also includes ε subunit – has 3’ to 5’ exonuclease activity – proofreading
• γ complex – multi-protein complex of ATPases – major job - clamp loader
• includes τ subunit – dimerize 2 core enzyme complexes – can replicate
  both strands coordinately
• β subunit – dimer that makes a donut shaped clamp – DNA in the hole – Pol
  doesn’t let go of DNA, therefore is highly processive

Question 7

What is the replisome?

Answer 7

REPLISOME – complex containing 2 DNA Pol III
holoenzymes, primosome and
DNA unwinding proteins
- one replisome at each replication fork

Question 8

What is the major polymerase for DNA replication in eukaryotes

Answer 8

DNA Pol III

Question 9

What is the function of DNA Pol I in prokaryotes?

Answer 9

DNA Pol I – 5’ to 3’ exonuclease – can degrade primer

• DNA polymerase activity – can fill in gaps (10 nts/sec)
• 3’ to 5’ exonuclease – proofreading
• Has two jobs – DNA replication and DNA repair

Question 10

How is prokaryotic DNA replication terminated?

Answer 10

Termination of replication -

• DNA replication ends at Ter
• Tus binds DNA at Ter – arrests replication when replisome arrives
• replisome disassembles

Question 11

How are the concatenated DNA daughter molecules separated?

Answer 11

Topoisomerases

Question 12

What are the phases of the cell cycle?

Answer 12

Four phases in the cell cycle
-G0
/G1 – “GAP”
- S phase – “synthesis” – DNA synthesis occurs here
replication all DNA – 1X ONLY
- G2 – “GAP”
- M – mitosis – chromosomes separate and cell division
occurs to make 2 daughter cells

Question 13

What is the function of DNA pol alpha?

Answer 13

α is primase – makes 10 nt RNA to bind and extends for

about 10 nt of DNA

Question 14

What is the function of DNA pol epsilon and delta?

Answer 14

• ε takes over and replicates the leading strand
• δ takes over and replicates the lagging strand

DNA Pol δ and ε are accurate, processive and have
3’ to 5’ exonuclease activity

Question 15

What is the function of DNA Pol γ?

Answer 15

– replication and repair of mitochondrial DNA

Question 16

What is licensing?

Answer 16

Licensing – mechanism to make sure DNA

replicates only once

Question 17

What is the preRC

Answer 17

preRC – preinitiation replication complex
– forms at ori
preRC = ORC (origin of replication complex)
+ replication licensing factors

ORC is analogous to DnaA in prokaryotes

Question 18

What is the replication licensing factor?

Answer 18

Replication licensing factors =

cdc6/cdc18/cdt1 complex + MCM complex

Question 19

What is MCM?

Answer 19

Major DNA helicase

Question 20

What are the steps in DNA licensing?

Answer 20

1 - Cell cycle is highly regulated by
kinases (Cdk)
2 - Cdc6 and cdt1 are phosphorylated
at the appropriate time and are destroyed
3 - DNA Pols are recruited
4 - Phosphorylated MCM unwinds DNA
5 - RPA – replication protein A binds ssDNA to stabilize
6 - Priming and DNA synthesis occurs