Ch. 20: DNA Replication

Question 1

Semiconservative replication

Answer 1

When a new double-stranded DNA molecule is formed, one strand will be from the original template, and one will be newly synthesized

Question 2

Who discovered semiconservative replication?

Answer 2

Meselson and Stahl

Question 3

Is DNA replication unidirectional or bidirectional?

Answer 3

Bidirectional

Question 4

Bidirectional DNA replication

Answer 4

The parent strands of DNA are antiparallel and can only be synthesized in the 5’ to 3’ direction, so the two new strands are synthesized in opposite directions

Question 5

Leading strand synthesis

Answer 5

Occurs in the 5’ to 3’ direction

Question 6

Lagging strand synthesis

Answer 6

5’ to 3’ direction, but in a discontinuous manner (Okazaki fragments)

Question 7

What seals together the Okazaki fragments?

Answer 7

DNA ligase

Question 8

What opens up the DNA helix?

Answer 8

Helicase

Question 9

Replication fork

Answer 9

The point at which the DNA helix is separated into two strands to allow for the replication of both strands

Question 10

DNA polymerase mechanism

Answer 10

Responsible for the bulk of DNA synthesis

Question 11

What does DNA polymerase do?

Answer 11

Catalyzes the addition of a deoxynucleotide (dNTP) to the 3’ end of the growing strand

Question 12

Klenow fragment

Answer 12

Contains polymerizing and editing modes without the 5’ to 3’ exonuclease activity

Question 13

Cleavage of DNA polymerase 1 results in what two fragments?

Answer 13

1. 5’ to 3’ exonuclease activity
2. Klenow fragment

Question 14

5’ to 3’ exonulcease activity

Answer 14

Makes is unsuitable for many applications

Question 15

What does the E. coli replication fork consist of?

Answer 15

1. Pol III
2. Helicase
3. Primase
4. Gyrase
5. Single-stranded DNA binding proteins

Question 16

Tethering both Pol III core complexes to helices by T subunits in the beta-clamp loading complex…

Answer 16

Ensures that the leading and lagging strand synthesis occur at a similar rate

Question 17

Clamp loader complex

Answer 17

Mediates the coordinated DNA synthesis on the leading and lagging strand templates by 2 Pol III core complexes

Question 18

Pol III Core

Answer 18

On the lagging strand template, it alternates between bound and unbound forms as each Okazaki fragment is made

Question 19

Termination Proteins (Prokaryotes)

Answer 19

To initiate replication, DNA synthesis at Oric must be separated. It is defined by sequence; several steps involve ATP binding or hydrolysis

Question 20

Termination region

Answer 20

DNA synthesis is initiated at Oric and proceeds bidirectionally until the DNA replication forks each reach halfway around the genome at this sequence

Question 21

Formation of the pre-replication complex (pre-RC)

Answer 21

Starts with the binding of CdC6, CdH, and two McM2-7 helices to the ORC

Question 22

When are the pre-RC’s formed?

Answer 22

During the G1 phase

Question 23

When does the activation of specific sites of the pre-RC happen?

Answer 23

During the S phase

Question 24

What does the pre-RC do?

Answer 24

It converts to a replisome progression complex (RPC) after the binding of additional proteins at the two replication forks.

Question 25

Short patch-repair (base excision repair)

Answer 25

Involves removal and replacement of a single nucleotide catalyzed by DNA polymerase and DNA lyase

Question 26

Long patch repair (base excision repair)

Answer 26

Involves the synthesis of up to 10 or more nucleotides using a strand displacement mechanism; Flap endonuclease (FEN) removes the displaced strand, leaving behind a single-strand nick that is sealed by DNA ligase

Question 27

Nucleotide excision repair

Answer 27

Initiated by recognition of the lesion by UvrAB complex; used for large lesions that distort the helical nature of DNA

Question 28

What causes nicks in the DNA in nucleotide excision repair?

Answer 28

UvrB and UvrC

Question 29

What does UvrD do in nucleotide excision repair?

Answer 29

Removes the polynucleotide containing the lesion, followed by gap repair using Pol I and ligase

Question 30

What does UvrABC exonuclease do in nucleotide excision base repair?

Answer 30

Scans for errors in the DNA

Question 31

DNA Photolyase (Direct Repair)

Answer 31

Photolyase converts thymine dimers to their normal structures via a 6 step mechanism

Question 32

6 Step Mechanism of DNA Photolyase

Answer 32

1. Absorption of lightt by the folate coenzyme (MTHF ) 2. Energy transfer from the MTHF* to FADH- to generate the excited state *FADH- 3. E- transfer from *FADH- to the cyclobutane dimer substrate 4. Radical reaction breaks the 1st bond 5. Radical reaction breaks 2nd bond 6. E- is transferred to FADH, and adjacent pyrimidines restored to normal structure

Question 33

MGMT (Direct repair)

Answer 33

The MGMT enzyme uses a suicide mechanism to remove the CH3 group from O6-methylguanine An active site cytosine residue displaces the methyl group to produce a homocysteine residue This modification inactivates the protein, which is then degraded to metabolize the homocysteine

Question 34

Repair of Single-Strand breaks in eukaryotes

Answer 34

The process of repairing a single-strand DNA break in eukaryotes is very similar to base excision repair and nucleotide excision repair mechanisms

Question 35

What is the difference in single-strand breaks in eukaryotes vs. prokaryotes?

Answer 35

Different repair proteins are required for short repair and long repair

Question 36

How is the gap fixed after long and short DNA repair (eukaryotes)?

Answer 36

It is sealed by one of several DNA ligases (Lig1 or Lig3)

Question 37

What are the two methods of repair for double-strand breaks in eukaryotes?

Answer 37

1. Homologous recombination 2. Nonhomologous end joining

Question 38

Non-homologous end joining

Answer 38

1. Does not require a DNA template strand

Question 39

Homologous recombination (Double-strand break repair)

Answer 39

In this repair method, a DNA template is used to fix the double strand break through homologous combination. This is a method used in CRISPR.