DNA Replication

Question 1

Function: DnaA

Answer 1

oriC recognition protein - bind at DnaA box next to oriC and melt DNA after oligomerization

Question 2

Function: DnaB

Answer 2

bacterial replicative helicase - unwind DNA at replication fork

Question 3

Function: DnaC

Answer 3

helicase (DnaB) loader - loads helicase (lol)

Question 4

Function: DnaG

Answer 4

bacterial primase - make RNA primer

Question 5

Prokaryotic Replication Initiation Steps

Answer 5

1) DnaA binds upstream oriC and oligomerizes
2) oligomerization melts oriC by making solenoidal supercoil
3) DnaC loads DnaB onto bubble
4) DnaC comes off

Question 6

Function: ORC

Answer 6

initiator protein, recognizes origin of replications

Question 7

Function: MCM2-7

Answer 7

eukaryotic replicative helicase - unwind DNA at replication fork

Question 8

Function: Cdc6

Answer 8

Co-initiator, helicase loader - helps MCM2-7 bind properly to ORC

Question 9

Function: Cdt1

Answer 9

Helicase loader - loads MCM2-7 onto ORC and origin of replication

Question 10

Function: Pol α

Answer 10

eukaryotic primase - make RNA primer

Question 11

Eukaryotic Replication Initiation Steps

Answer 11

1) ORC binds to origin of replication
2) With ATP, Cdc6 binds (coinitiator)
3) With ATP, Cdt1 loads MCM2-7 to ORC
4) with ATP, Cdc45 causes maturation of MCM2-7

Note: MCM2-7 is loaded as dodecamer, splits into hexameric rings on maturation

Question 12

Function: HDA

Answer 12

hydrolyzes ATP-DnaA to prevent oligomerization, stops reinitiation

Question 13

Function: CLP

Answer 13

Converts ADP-DnaA to Apo-DnaA

Question 14

Function: SeqA

Answer 14

binds hemimethylated DNA post replication initation to prevent origin refiring

Question 15

Bacterial Replication Initiation Regulation

Answer 15

Hda dephosphorylates ATP-DnaA, CLP replaces ADP-DnaA with Apo-DnaA, Apo-DnaA can get recycled to ATP-DnaA

SeqA can bind hemimethylated DNA

Question 16

Eukaryotic Replication Initiation Regulation

Answer 16

Post-Translational Modifications
Proteolysis
Nuclear Export

Question 17

Function: The CMG

Answer 17

Elongation Helicase

Question 18

Function: Pol III

Answer 18

Bacteria DNA Polymerase

Question 19

Function: Pol ε, Pol δ

Answer 19

Eukaryotic DNA Polymerase

Question 20

CMG vs DnaB Difference in Directionality

Answer 20

DnaB - 5’ -> 3’ movement on lagging strand template

CMG - 3’ -> 5’ movement on leading strand template

Question 21

Why so many Polymerase families?

Answer 21

Many functions to perform, such as priming, leading/lagging strand synthesis, repair

Question 22

Function: Translesion (TLS) polymerases

Answer 22

cannot fix damage, has to just make an error and move on

Question 23

Polymerase Fidelity

Answer 23

“Steric gate”
Snug active site - sense shape complimentarity
Disfavor rNTP binding
Exclude wobble/mispairs

Question 24

Function: β protein

Answer 24

Bacterial clamp

Question 25

Function: PCNA

Answer 25

Eukaryotic clamp

Question 26

Function: RFC

Answer 26

Eukaryotic Clamp Loader

Question 27

Function: the τ complex

Answer 27

Bacterial clamp loader

Question 28

Function: Ssb

Answer 28

Bacterial single strand binding protein

Question 29

Function: RPA

Answer 29

Eukaryotic single strand binding protein

Question 30

What does the clamp do during replication?

Answer 30

Increase processivity of DNA pol

Question 31

Clamp Loader Mechanism

Answer 31

1) ATP binding opens clamps
2) 3’ end of primer enters clamp loader
3) ATP hydrolysis closes clamp closes,

Question 32

Leading/Lagging Strand Linking

Answer 32

Bacteria: clamp loader connects helicase + polymerase
Eukaryote: Helicase binds pol + scaffold factor

Question 33

Function: Ctf4

Answer 33

Scaffold factor that tethers lagging strand to helicase

Question 34

How do cells deal with Okazaki Fragments?

Answer 34

3’->5’ degradation
RNase H cleaves primer, extend with Pol, ligate
Dna2/FenI mechanism through flap formation

Question 35

RNase H Mechanism

Answer 35

2-metal ion mechanism

Mg ion goes into high energy state when nucleotide binds

Question 36

DNA Ligase mechanism

Answer 36

Charge ligase with AMP through ATP hydrolysis
AMP transferred to 5’ phosphate
3’ OH of acceptor will attack 5’ phosphate

Ligase encircles DNA

Question 37

Premature Termination Mechanisms

Answer 37

Protein mediated block, Fork collision, superhelical strain

Question 38

What are catenanes?

Answer 38

interlocking rings of DNA that cannot be separated without breaking covalent bonds, formed during replication/transcription

Need to unlink

Question 39

Type I Topo vs Type II Topo

Answer 39

Type 1 cuts 1 strand, type 2 cuts 2 strands

Both go through tyrosyl-DNA intermediate

Question 40

Type 1B Topo Mechanism

Answer 40

Nick-and-Swivel

Too lazy to explain

Question 41

Topo IIA Mechanism

Answer 41

1) ATP binds and traps gate segment
2) cleaves and pulls another DNA strand through
3) Releases ADP and can accept another G segment

processive, can unlink catenanes

Question 42

What type of supercoils do histones stabilize?

Answer 42

Stabilize solenoidal supercoils, negative

Question 43

Histone Modifications for Replication

Answer 43

H4K20me2 -> ORC

Question 44

Histone Segregation

Answer 44

Mcm2 has H3-H4 chaperdone domain that aids apportionment.

Equal apportionment of old and new nucleosomes

Question 45

T/F. Initiation, elongation and termination factors are all broadly conserved.

Answer 45

False, initiation machineries are broadly conserved, but elongation and termination factors are not