Rodgers 2-5 to 2-10

Question 1

Where does energy come from to produce the energetically unfavorable addition of complements to a growing DNA strand

Answer 1

Hydrolysis of PPi from incoming NTPs

Question 2

How large is E. Coli’s genome

Answer 2

~4.6 million b.p.’s

Question 3

What 2 characteristics define the OriC region of E. coli

Answer 3

~3 A-T-rich-13bp tandem repeat segments, and ~5 9bp DnaA boxes

Question 4

How does DnaA affect the DNA Lk?

Answer 4

Positively supercoils (increases Lk)

Question 5

What are the domains of DnaA?

Answer 5

Helix-turn-helix motif, ATPase domain

Question 6

How does DnaA interact with DNA?

Answer 6

One helix provides specificity by sitting in the major groove, One helix provides affinity by interacting with the neg. charge backbone

Question 7

What 2 characteristics define yeast replication origin sites

Answer 7

Autonomously Replicating Sequences (ARS) Consensus Sequences (ACS), B-elements (containing DNA Unwinding Elements- DUEs)

Question 8

What is the eukaryotic equivalent of DnaA?

Answer 8

ORC

Question 9

How is ORC different from DnaA

Answer 9

Made of 6 different proteins

Question 10

How do chromosomes appear during replication

Answer 10

Stretched out

Question 11

How many replicons in human genome

Answer 11

~50,000

Question 12

What are groups of clustered replicons called

Answer 12

foci

Question 13

How are fission yeast origin sites different from budding yeast?

Answer 13

They have Origin Binding Recognition Sites (OBRs) that are very large and vary variable

Question 14

What is the function of Cdc6 and Cdt1in yeast

Answer 14

They work with ORC to recruit MCM’s (mini-chromosome maintenance helicases)

Question 15

Why are some replisomes not activated in Eukaryotes

Answer 15

presence of CpG islands, methylation, or other transcription-regulating machinery - sometimes this is an environmental effect

Question 16

What is the main difference between MCM and DnaB helicase

Answer 16

MCM goes around double stranded DNA, while DnaB is toroid hexamer around one strand

Question 17

What are the 2 distinct domains are found in Orc1 of ORC?

Answer 17

winged helix (identifies bp’s in the major groove) , and AAA+ ATPase (interacts with minor groove)

Question 18

Why is DNA deformed from its original bDNA helix when bound by DNA recognizing proteins

Answer 18

Because the protein creates an induced-fit conformation on the DNA (more energetically conducive to binding)

Question 19

What shape does the ORC make on DNA? What probable purpose is there for this?

Answer 19

a ‘kink,’ perhaps to help load the helicase

Question 20

What is the ‘processivity number’

Answer 20

the number of base pairs a polymerase can traverse before falling off

Question 21

What loads the helicase to the DNA

Answer 21

an ATPase, like ORC or DnaA

Question 22

What increases processivity for polymerases

Answer 22

the bonding interaction with a clamp (helicase)

Question 23

What 2 things comprises the checklist for what must occur before replication starts

Answer 23

1. Cell must be large enough 2. Must not have just opened the DNA for replication (DnaA-ATP:DnaA-ADP ratio of 1)

Question 24

What 3 things prevent re-initiating replication in E. Coli

Answer 24

1. SeqA binds at OriC, 2. DatA locus soaks up free DnaA proteins near the OriC, 3. Less DnaA-ATP is available

Question 25

What is the action of SeqA in E. coli

Answer 25

SeqA binds hemi-Me GATC i(Dam sites) n the OriC to prevent DnaA binding

Question 26

Which is more abundant in the cell: ATP or ADP?

Answer 26

ATP

Question 27

Why is cell size important for regulating initiation of replication in E. coli?

Answer 27

faster growth means faster protein production, means quicker resumption of 1:1 ratio of DnaA-ATP/ADP - may re-initiate replication faster

Question 28

Who was the first to characterize Ploymerases of E.coli?

Answer 28

Arthur Kornberg

Question 29

What is the most abundant , most well-studied polymerase in E. coli?

Answer 29

Pol. I

Question 30

Which polymerase of E. coli does the most work? Why?

Answer 30

Pol. III- it is the most processsive

Question 31

Where are polymerases' active sites located on the complex?

Answer 31

In the 'palm' domain

Question 32

What do the fingers and thumb domain do in Polymerase?

Answer 32

Interact with the backbone to generate affinity to the DNA

Question 33

Which polymerase does most of the work in replication for Eukaryotes?

Answer 33

Pol. alpha

Question 34

What are the features of all polymerase active sites?

Answer 34

central beta sheets, flanking helices, and catalytic aspartate residues

Question 35

What is special about aspartate? What function does it have?

Answer 35

Neg charge. It coordinates divalent ions (Mg) bonding with DNA backbone

Question 36

What 3 things do the divalent metal ions do during polymerization?

Answer 36

1. Encourage the ionized (deprotonated) form of the 3'OH to make it act as a better nucleophile 2. Coordinates the in-line exit of the PPi leaving group (LG) 3. Stabilizes the charge of the PPi LG

Question 37

What makes the core of Pol. III in E. coli?

Answer 37

Three enzymes (alpha, epsilon, theta)

Question 38

What process makes the Pol III holoenzyme processive

Answer 38

the atp-bound gamma complex attracts the beta clamp subunit (forces open conformation when binds), then when DNA enters the clamp, hydrolysis is stimulated in gamma which closes beta. gamma and beta lose affinity for one another.