Chapter 13 - DNA Replication

Question 1

Initiation

Answer 1

1) Parental strands are separated
2) The ssDNA are stabilized
3) A replication bubble is

Question 2

Elongation

Answer 2

1) Assembly of replisome, a multiprotein structure, that undertakes DNA synthesis at the replication fork
2) Replisome moves along the DNA
3) Parental strands unwinds
4) Daughter strands are synthesized.

Question 3

Termination

Answer 3

The joining of the replicons after which the duplicated

chromosomes are separated.

Question 4

Conditional Lethal

Answer 4

Replication mutation. Example: Temperature sensitivity.
Because inability to replicate DNA is fatal, must generate mutants that are able to replicate only under permissive conditions but die otherwise.

Question 5

Direction of DNA synthesis

Answer 5

5’-3’

Question 6

Precursor for DNA synthesis

Answer 6

Nucleoside triphosphate that loses the terminal two phosphate groups in the reaction.

Question 7

DNA synthesis is required for

Answer 7

Semi-conservative DNA replication and DNA-repair

Question 8

Replicase

Answer 8

DNAP III. One bacterial DNA polymerase undertakes semiconservative replication

Question 9

DNAP α

Answer 9

High-fidelity replicase in eukaryotes. Nuclear replication.

Question 10

DNAP δ

Answer 10

High-fidelity replicase in eukaryotes. Lagging strand.

Question 11

DNAP ε

Answer 11

High-fidelity replicase in eukaryotes. Leading strand.

Question 12

DNAP γ

Answer 12

High-fidelity replicase in eukaryotes. Mitochrondrial replication.

Question 13

E. coli DNA polymerase I

Answer 13

Unique 5’ and 3’ exonuclease activity.

A 103 kD polypeptide that can be cleaved into a large subunit (68 kD) and a small subunit (35 kD).

Question 14

DNAP I Large Subunit

Answer 14

Klenow Fragment

1. Polymerase
2. 3’-5’ Exonuclease

Question 15

DNAP I Small Subunit

Answer 15

5’-3’ Exonuclease

Question 16

The fidelity of replication relies on

Answer 16

Specificity of base pairing.

Question 17

Two classes of DNA polymerase-induced errors:

Answer 17

1. Substitutions

2. Frameshifts

Question 18

Correction of a substitution error

Answer 18

Proofreading scrutinizes newly formed base pairs.

Question 19

Correction of a frameshift error

Answer 19

Reduced with greater enzyme processivity.

Question 20

Processitivity

Answer 20

The tendency of the enzyme cycle on and off the DNA strand/template.

Question 21

Fidelity mechanism for high-fidelity DNAP

Answer 21

The geometry of the base-pairing. Improper fitting stalls the DNAP. Moves the DNAP back to remove the mismatched base.

Question 22

Three domains of many DNAP

Answer 22

Form a large cleft that resemble a right hand.
1. The “palm” includes a conserved sequence motifs that
constitute the active site.
2. The “fingers” (variable) involved in positioning the DNA.
3. The “thumb” (variable) binds DNA and important for processivity.

Question 23

DNAP nuclease activity

Answer 23

Resides in a completely different domain from large cleft/hand that positions DNA.

Question 24

What exposes the template to an incoming nucleotide while held in the DNAP “hand”

Answer 24

A sharp turn in the parental strand exposes the template to the incoming nucleotide.
The 3’end to which the nucleotide is being added is anchored by the fingers & palm.

Question 25

Contacts between DNAP "hand" and DNA

Answer 25

Via phosphodiester bond

Question 26

Rotation of the DNAP during DNA replication

Answer 26

The fingers rotate 60-degrees while the thumb rotates 8-degrees towards the palm. Rotational changes is cyclic.

Question 27

Is the lagging strand DNA replication continuous or discontinuous?

Answer 27

The lagging strand is synthesized in sequential fragments (Okazaki fragments) or discontinuously in the 5’-3’ direction and subsequently joined/ligated together. Hence it is semidiscontinuous replication.

Question 28

What is required to generate and maintain ssDNA for replication?

Answer 28

1. A helicase to separate/unwind the strands of DNA using energy provided by hydrolysis of ATP. 2. A single-stranded binding protein (SSB) is required to maintain the separated strands.

Question 29

What do all DNAP require ?

Answer 29

A 3’-OH priming end to initiate DNA synthesis or a primer.

Question 30

Methods to provide the free 3'-OH DNAP require to initiate DNA synthesis?

Answer 30

- RNA primer - a nick in DNA - a priming protein

Question 31

How many times is the RNA primer uses in leading versus lagging strand?

Answer 31

- One (1) time in Leading Strand | - Multiple times in Lagging Strand

Question 32

Primase

Answer 32

Synthesizes the RNA chain that provides the priming end

Question 33

What does priming of replication (starting DNA synthesis) on double stranded DNA always require?

Answer 33

A replicase (DNA Pol), helicase (DnaB), SSB, and primase (DnaG).

Question 34

E. Coli priming reactions

Answer 34

1. The oriC system - involves the DnaG primase at the fork. 2. The φX system - requires a primosome to restart replication at a damaged fork.

Question 35

How many DNAP catalytic units are required to synthesize the leading and lagging strands?

Answer 35

Two dimeric DNAP catalytic units are required: - One enzyme unit elongates the leading strand processively in the same direction. - The other enzyme unit must move in the reverse direction to synthesize an Okazaki fragment backwards

Question 36

Each monomeric unit of the E. Coli replicase (DNAP III) dimeric structure contains:

Answer 36

- A catalytic core that includes the α, ε, and θ subunits. - A dimerization subunit or τ that links 2 catalytic cores. - A processivity component that includes the β-ring clamp (holds the catalytic core on the template) and γ complex clamp loader.

Question 37

Catalytic core includes

Answer 37

α, ε, and θ subunits

Question 38

T-unit

Answer 38

Dimerization subunit - links 2 catalytic cores.

Question 39

Processivity component

Answer 39

Includes the β-ring clamp (holds the catalytic core on the template) and γ complex clamp loader.

Question 40

Clamp loader

Answer 40

Uses ATP hydrolysis to open the β-ring/bind β-subunits to a template. Binding DNA changes the conformation of the β-subunits. Enhances the recruitment of the catalytic core. Brings the enzyme core to the DNA. Dimerization component binds the 2 core enzyme. Each enzyme core synthesizes one of two new strands of DNA. Clamp loader dissociates (but remains close) once the clamp is positioned on the DNA. Cyclic process at the lagging strand.

Question 41

How does the β-ring clamp associate with DNA?

Answer 41

The β-subunit of DNA Pol III holoenzyme is a head-to-tail dimer. Forms a ring completely around the DNA. The β-ring clamp “skates” along the DNA. No bonds. Associates with DNA via the intermediate water molecules between the clamp and the DNA.

Question 42

Helicase DnaB

Answer 42

Responsible for interacting with the primase DnaG to initiate each Okazaki fragment. Contacts the τ subunits of the clamp loader to connect the helicase, primase, and the catalytic core.

Question 43

Trombone model

Answer 43

Synthesis of the leading strand creates a loop of ssDNA on the lagging strand. The enzyme complex on the lagging strand that “pulls” the ssDNA through the clamp while synthesizing the new strand.

Question 44

Low-fidelity DNAP are also known as

Answer 44

Error-prone polymerases.

Question 45

How many subunits do replicases have?

Answer 45

Four (4) subunits (tetramers) for catalysis, priming, processivity, and proofreading.

Question 46

DnaB/MCM complex

Answer 46

Helicase

Question 47

DnaC/Cdc6

Answer 47

Loading helicase/primase

Question 48

SSB/RPA

Answer 48

Single-strand maintenance

Question 49

DnaG/DNAPα/primase

Answer 49

Priming

Question 50

τ

Answer 50

Holoenzyme dimerization

Question 51

Pol I/FEN1

Answer 51

RNA removal

Question 52

Ligase/Ligase 1

Answer 52

Ligation

Question 53

Pol III core/Pol ε/Pol δ

Answer 53

Catalysis

Question 54

Pol ε

Answer 54

Elongates the leading strand

Question 55

Pol δ

Answer 55

Elongates the lagging strand

Question 56

β/PCNA

Answer 56

Processivity

Question 57

Three different DNAP make up eukaryotic replication fork

Answer 57

Pol α/primase (priming) DnaB/MCM (helicase unwinding) β/PCNA (processivity)

Question 58

Which DNA polymerase expresses a nuclease activity (E. Coli or mammal)?

Answer 58

E. coli DNAP I express nuclease activity to remove a primer.

Question 59

How do mammalian DNAP remove a primer?

Answer 59

1. RNase H makes an endonucleolytic cleavage to generate a “flap”. 2. FEN1 is a 5’-3’ exonuclease that recognizes and cleaves the RNA primer “flap”.

Question 60

Two options when replication fork stalls at a damaged base or nick in DNA

Answer 60

Lesion bypass OR recombination.

Question 61

Lesion bypass

Answer 61

Occurs during replication with an error-prone DNAP on a template that contains a damaged base. Error-prone DNAP can incorporate a noncomplementary base into the daughter strand. The mismatch is repaired at a later time.

Question 62

Primosome

Answer 62

Required to restart a stalled replication fork after the DNA has been repaired. Binds and functions to reload a new DnaB to the reinitiate replication.

Question 63

Recombination (after replication fork stalls)

Answer 63

The damaged sequence is excised. The complement of the other daughter duplex crosses over to repair the gap. Allows replication to continue.

Question 64

Termination of replication in E. coli

Answer 64

Called ter site. ~23 bps Function is unidirectional. E. coli have 2 cluster of 5 ter sites. Each set allows the replication fork into the termination region, but does not allow it out the other side = "replication fork trap". The linear eukaryotic chromosomes most likely use a modified form of the ligation process.