8.8.16 Lecture

Question 1

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

Answer 1

A-T

C-G

Question 2

Describe the structure of DNA.

Answer 2

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.

Question 3

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

Answer 3

1. Template strand - strand used to create a copy

2. Primer strand - begins the process of DNA synthesis

Question 4

DNA polymerase requires a ___.

Answer 4

3’-OH

Question 5

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

Answer 5

The 5’ triphosphate of the incoming deoxyribonucleoside triphosphate.

Question 6

DNA is always synthesized in the ___ direction.

Answer 6

5’ to 3’

Question 7

Describe the two strands formed in DNA synthesis.

Answer 7

1. Leading strand: continuous 5’ to 3’ synthesis

2. Lagging strand: Okazaki fragments in the 5’ to 3’ direction

Question 8

DNA replication is bi-directional. Describe this process.

Answer 8

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.

Question 9

What are the four important characteristics governing nucleic acid synthesis?

Answer 9

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.

Question 10

What are the six steps of DNA replication?

Answer 10

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

Question 11

Where does initiation begin?

Answer 11

At origins of replication

Question 12

Describe the process of initiation of DNA replication.

Answer 12

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.

Question 13

___ is an allosteric motor protein that unwinds DNA.

Answer 13

Helicase

Question 14

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

Answer 14

1 molecule of ATP

Question 15

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

Answer 15

SSBs prevent reannealing without preventing base pairing.

Question 16

What happens without SSBs?

Answer 16

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

Question 17

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

Answer 17

Cooperatively.

Question 18

DNA synthesis requires an ___.

Answer 18

RNA primer

Question 19

What does the sliding clamp do?

Answer 19

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

Question 20

How does the sliding clamp function?

Answer 20

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.

Question 21

Describe the process of addition and removal of RNA primer.

Answer 21

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.

Question 22

What does ligase do?

Answer 22

Ligase synthesizes the phosphodiester bond that links Okazaki fragments.

Question 23

How does ligase work?

Answer 23

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

Question 24

DNA polymerase ___ as it synthesizes DNA.

Answer 24

Proofreads

Question 25

How does DNA polymerase proofread as it synthesizes DNA?

Answer 25

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

Question 26

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

Answer 26

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

Question 27

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

Answer 27

DNA polymerase

Question 28

Which strand is more dependent on SSD binding proteins?

Answer 28

Lagging

Question 29

Why does the lagging strand have to fold back?

Answer 29

The replication proteins act together as one large multienzyme complex.

Question 30

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

Answer 30

Torsional stress and superhelicity

Question 31

What reliever supercoils and untangles DNA during replication?

Answer 31

Topoisomerase

Question 32

What does DNA topoisomerase I do?

Answer 32

Relieves supercoiling ahead of the replication fork

Question 33

How does DNA topoisomerase I function?

Answer 33

1. DNA topoisomerase I has tyrosine at its active site. It covalently attaches to a DNA phosphate, breaking a phosphodiester linkage in one DNA strand. 2. The two ends of the DNA double helix can now rotate relative to each other, relieving accumulated strain. 3. The original phosphodiester bond energy is stored in the phophotyrosine linkage, making the reaction reversible. 4. Spontaneous reformation of the phosphodiester bond regenerates both the DNA helix and the DNA topoisomerase.

Question 34

What does DNA topoisomerase II do?

Answer 34

Untangles DNA after it is replicated.

Question 35

How does DNA topoisomerase II function?

Answer 35

1. Two circular DNA double helices are interlocked. Type II DNA topoisomerase makes a reversible covalent attachment to opposite strands, interrupting one helix and forming a protein gate. 3. The topoisomerase gate opens and shuts to let a second helix pass. 4. Reversal of the covalent attachment of the topoisomerase restores an intact double helix.

Question 36

Why are DNA topoisomerase II inhibitors effective cancer drugs?

Answer 36

Increase stability of topo-DNA cleavage complexes, leading to high levels of transient DNA breaks. These become permanent when replication machinery or helicases attempt to traverse the DNA. This leads to cell death.

Question 37

What is the mechanism that ensures each origin of replication is activated only once per cycle?

Answer 37

1. In G1, origin recognition complexes (ORCs) bind to the origin, along with the helicase to form a pre-replicative complex. 2. In S, the helicase and ORC are phosphorylated by kinases, leading to displacement of ORC and recruitment of DNA polymerase and other replication protein. The ORC rebinds and synthesis begins. 3. In G2, the replication is completed.

Question 38

Describe how patterns of histone modification can be inherited.

Answer 38

H3-H4 tetramers are distributed to the daughter DNA randomly, with roughly equal numbers inherited by each daughter. H2A-H2B dimers are released from the DNA as the replication fork passes. Reader-writer complexes recognize the same modifications they catalyze and fill in the blanks.

Question 39

What are the three major ways eukaryotic chromosomal DNA replication differs from prokaryotic DNA replication?

Answer 39

1. Compartmentalized within the nucleus, partitioning it from the site of synthesis of replication proteins and precursors and from extracellular stimuli that may trigger initiation of synthesis. 2. Begins at multiple origins, activated throughout S-phase in a precise and temporally regulated manner 3. The nucleosomal proteins must be duplicated along with the DNA to maintain proper chromosomal organization.

Question 40

What is telomerase?

Answer 40

A riboprotein containing an RNA template for synthesizing the G-rich telomere sequence

Question 41

Telomerase is primarily expressed in what three types of cells?

Answer 41

Germ, stem, and cancer cells

Question 42

Why is telomerase needed?

Answer 42

The 3' end of the template DNA strand has no room for a primase to synthesize a primer.

Question 43

What does telomerase do?

Answer 43

Elongates the 3' end

Question 44

Telomere shortening can trigger...

Answer 44

...replicative senescence in human cells that don't express telomerase.

Question 45

How does trinucleotide repeat expansion occur?

Answer 45

DNA replication slippage - DNA polymerase pauses while replicating through the triple-repeat tract and transiently dissociates. During reassociation, a misalignment between the template and primer strands would result in unpaired repeat sequences either on the template or on the new strand. If the mismatch is not repaired, the unpaired sequence on the primer strand will cause expansion of the repeat tract following the next round of DNA replication. Note that contraction can also occur when the misalignment happens on the template strand.