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1

What are the correct Watson-Crick base pairings for DNA?

A-T
C-G

2

Describe the structure of DNA.

DNA is a double-stranded helix. Each strand runs from the 5' to the 3' end. The strands are aligned in an anti-parallel fashion.

3

What are the names and roles of the two strands in DNA replication?

1. Template strand - strand used to create a copy
2. Primer strand - begins the process of DNA synthesis

4

DNA polymerase requires a ___.

3'-OH

5

What engages in a nucleophilic attack on the 3' end of the primer strand?

The 5' triphosphate of the incoming deoxyribonucleoside triphosphate.

6

DNA is always synthesized in the ___ direction.

5' to 3'

7

Describe the two strands formed in DNA synthesis.

1. Leading strand: continuous 5' to 3' synthesis
2. Lagging strand: Okazaki fragments in the 5' to 3' direction

8

DNA replication is bi-directional. Describe this process.

1. Replication begins at an origin of replication.
2. The DNA helix is opened locally.
3. RNA primer is synthesized on each strand.
4. Leading-strand DNA synthesis begins.
5. RNA primers start lagging strand synthesis
In total, this leads to 2 forks from each origin - each with a leading strand and a lagging strand.

9

What are the four important characteristics governing nucleic acid synthesis?

1. Pre-existing nucleic acid strand is copied by the rules of Watson-Crick base pairing
2. Nucleic acid strands grow in the 5' to 3' direction only
3. Polymerases synthesize nucleic acids.
4. Duplex DNA synthesis requires a special growing fork because the strands are antiparallel.

10

What are the six steps of DNA replication?

1. Initiation
2. Unwinding
3. Priming
4. Unidirectional fork movement
5. Untangling
6. Termination

11

Where does initiation begin?

At origins of replication

12

Describe the process of initiation of DNA replication.

1. Initiator proteins bind to the origin and destabilize the AT-rich sequence. These proteins bind to and bend the DNA to loosen the double strand tightness.
2. DNA helicase binds to the helicase-loading protein and is loaded onto the DNA. When activated, helicases unzip the protein.
3. DNA primase is loaded (synthesizes RNA)
4. RNA primer synthesis enables DNA polymerase to start leading-strand synthesis.
5. Loading of 2 additional DNA polymerases allows lagging-strand synthesis to begin.
6. This process results in two replication forks moving in opposite directions.

13

___ is an allosteric motor protein that unwinds DNA.

Helicase

14

How much energy is needed for helicase to unzip one nucleotide?

1 molecule of ATP

15

What do single-strand binding proteins (SSBs) do?

SSBs prevent reannealing without preventing base pairing.

16

What happens without SSBs?

Single-stranded regions of DNA template can pair with one another, blocking replication.

17

SSBs bind ___ and straighten the backbone of the DNA chain while leaving the bases open and accessible.

Cooperatively.

18

DNA synthesis requires an ___.

RNA primer

19

What does the sliding clamp do?

A regulated sliding clamp permits processive DNA synthesis on the leading strand and rapid reassembly of the replication complex on the lagging strand.

20

How does the sliding clamp function?

1. A clamp loader binds to the sliding clamp and opens it using ATP.
2. The sliding clamp slips around the DNA.
3. ATP hydrolysis locks the sliding clamp around DNA and releases the clamp loader.
4. DNA polymerase binds to the sliding clamp.

21

Describe the process of addition and removal of RNA primer.

1. DNA primase synthesizes a new RNA primer.
2. DNA polymerase adds to the new RNA primer to start a new Okazaki fragment.
3. DNA polymerase finishes the DNA fragment.
4. DNA polymerase bumps into the old primer, activating RNAse, which erases the primer and replaces it with DNA.
5. DNA ligase seals the nick and joins the new fragment to the growing chain.

22

What does ligase do?

Ligase synthesizes the phosphodiester bond that links Okazaki fragments.

23

How does ligase work?

Ligase uses ATP to add a phosphate group to the 5' P. The 3' OH bonds to this new P. AMP is released.

24

DNA polymerase ___ as it synthesizes DNA.

Proofreads

25

How does DNA polymerase proofread as it synthesizes DNA?

3' to 5' exonuclease activity attached to DNA polymerase chews back to create a base-paired 3'-OH end on the primer strand.

26

What three things give rise to high-fidelity DNA synthesis?

1. 5' to 3' polymerization
2. 3' to 5' exonucleolytic proofreading
3. Strand-directed mismatch repair

27

Germline mutations affecting the ___ proofreading domains predispose people to colorectal adenomas and carcinomas.

DNA polymerase

28

Which strand is more dependent on SSD binding proteins?

Lagging

29

Why does the lagging strand have to fold back?

The replication proteins act together as one large multienzyme complex.

30

What are the two major winding problems that occur during DNA replication?

Torsional stress and superhelicity