Chapter 14

Question 1

1. Understand how the 20th century can be divided into “quarters” with each quarter leading to an important breakthrough in our understanding of genetics. What happened in 1900? What happened in the middle of the century? What happened in 2000?

Answer 1

1900 - Mendel rediscovered, figuring out that gene were on chromosome.
1950 - race to solve structure of DNA. Watson and Crick.
2000 - sequenced human genome

Question 2

Why were proteins initially believed to be the “genetic determinants”? What was known about DNA prior to 1900?

Answer 2

by the 1940s scientists knew that chromosomes carried hereditary material and consisted of DNA and protein. Most researchers thought protein was the genetic material because; proteins were macromolecules with great heterogeneity and functional specificity. Little was known about nucleic acids. The physical and chemical properties of DNA seemed too uniform to account for the multitude of inherited traits.
Not much about the DNA was known prior to 1900.

Question 3

Know the contributions of the following people in the identification of DNA as the material responsible for the transmission of genetic information and be able to describe and analyze the experiment that led to their contribution
- Frederick Griffith

Answer 3

“Transforming principle” – allowed change of nonvirulent R strain(benign) bacteria into virulent S strain bacteria.

Question 4

Know the contributions of the following people in the identification of DNA as the material responsible for the transmission of genetic information and be able to describe and analyze the experiment that led to their contribution:
- Avery, MacLeod, and McCarty

Answer 4

Their experiment showed that DNA is the informational component transferred during transformation.

Question 5

Know the contributions of the following people in the identification of DNA as the material responsible for the transmission of genetic information and be able to describe and analyze the experiment that led to their contribution:
- Alfred Hershey and Martha Chase

Answer 5

Hershey and Chase experiment using bacteriophage. 
Radiolabeled S35(labeling protein) and P32(DNA)

• Showed DNA contains genetic material.

Question 6

Transformation?

Answer 6

In Avery, Macleod, McCarthy, transformation occurred from R strain→S strain & found DNA was the informational component transferred during transformation

Question 7

Bacteriophage?

Answer 7

virus that infects bacteria

Question 8

What were Erwin Chargoff’s contributions to understanding DNA? What did Chargoff’s rules state?

Answer 8

• studying DNA from a chemical level
• reported that DNA composition varies from one species to the next but no matter what species he found the
  %A ≈ %T & %G ≈ %C.
• Chargoff’s rules: %A roughly equal to % T, %G roughly equal to %C

Question 9

Discuss the contributions of the following individuals in terms of determining the structure of DNA.

• Rosalind Franklin and Maurice Wilkins
• Francis Crick and James Watson

Answer 9

Franklin - X ray diffraction photograph of DNA suggesting DNA is double helix structure. Suggested the diameter of DNA molecule, and suggested that phosphates were on the backbone.

Watson and Crick took Franklin’s data and created DNA model

Question 10

7. Describe the double helical structure of DNA. How did Franklin’s X-ray crystallographic images contribute to our understanding of the physical structure of DNA? What insights did her work give Watson and Crick regarding the structure of DNA? How did the understanding of the structure of DNA explain Chargoff’s rules?

Answer 10

• Sugar phosphate backbone
• Double helix that has anti parallel strand.
  Franklins X-ray image
  Double helical structure of DNA: (what Watson & Crick published)
  1- DNA is a double helix (2 DNA strands in an anti-parallel orientation)
  2- 2 strands held together by H-bonding that’s very specific between A-T & G-C.
  3- Sugar-phosphate backbone is on the outside with the nitrogenous bases pointing inward to base pair with each other
  4- Purine(2 ring structure. A & G) always base paired with a pyrimidine(one ring structure C&T) making diameter perfect (2nm)
  5- 2 strands are complementary to one another, not identical

Question 11

a. major groove

b. minor groove

Answer 11

The major groove occurs where the backbones are far apart, the minor groove occurs where they are close together.

Question 12

c. Antiparallel

Answer 12

One strand 3’ to 5’ and other strand 5’ to 3’

Question 13

d. complementary strands of DNA

Answer 13

two complementary strands in DNA where nucleotides base pairs with complementary base pairing.

Question 14

e. 3’ end

f. 5’ end

Answer 14

Carbon 3, attached to -OH (hydroxyl) group

Carbon 5, attached to phosphate

Question 15

g. base-pairing

Answer 15

hydrogen bond between Purines (A&G) and Pyrimidines (T&C)

Question 16

How did Watson and Crick’s model of the double stranded DNA molecule provide a clue for how genetic information is copied in the cell?

Answer 16

realized that these pairing rules meant that either strand contained all the information necessary to make a new copy of the entire molecule, and that the aperiodic order of bases might provide a “genetic code”.

Question 17

Is DNA replication conservative, semi-conservative, or dispersive? Whose experiments led to the answer to this question?

Answer 17

Semi-conservative. Meselson & Stall’s experiment

Question 18

What is a “replication bubble” and a “replication fork”? What is an origin of replication?

Answer 18

Replication bubble: space between two separated strand of DNA in prokaryotes while replicating.
Replication fork: direction of replication. Replication is bidirectional(two replication fork)
Origin of replication: where first separation of DNA strand start.

Question 19

Understand how replication proceeds in a bidirectional direction from the origin resulting in two replication forks.
How does the number of origins in prokaryotic and eukaryotic cells differ?

Answer 19

There is one origin in prokaryotes and multiple origins in eukaryotes.

Question 20

Understand how the sequence of the AT rich region of the origin of replication is important in terms of the initial opening of the helix at the origin. What does helicase do once this region is opened?

Answer 20

DnaA complex binds and wraps around 9 bp repeats DNA complex. This creates tension at AT-rich regions of the origin of replication (A-T has 2 hydrogen bonds compared to G-C with 3 hydrogen bonds) opens up as a result. SSBP(single-stranded binding protein) bind to each strand and keep them from snapping back together. Two DnaC help load two DnaB/helicase onto each replication fork. Two DnaC complex leaves.

Question 21

Know the molecular mechanism of DNA replication. Where does the energy come from to make a phosphodiester bond? Why is magnesium required? Which end of the growing strand can you add the new nucleotide? Why?

Answer 21

What does DNA polymerase need to “do it’s job”?

• Anti-parallel template
• dNTP’s (nucleotides)
• Mg2+
• 3’ OH

Where energy comes from: every nucleotide comes as comes with nucleotide triphosphate. Breaking off 2 phosphates(pyrophosphate- beta and gamma phosphate) release free energy

Question 22

13. Understand the contribution of the following molecules in the process of replication:
    DnaA

Answer 22

what binds to 9 base pair repeats and pops up A-T rich region(13 bp repeats)

Question 23

contribution of the following molecules in the process of replication:
Helicase (DnaB)

Answer 23

Unwinds DNA double helix at replication forks

Question 24

contribution of the following molecules in the process of replication:
single-strand binding protein

Answer 24

prevent strands from being digested or closing before the replication

Question 25

contribution of the following molecules in the process of replication: DNA primase

Answer 25

Create RNA primers at 5’ end of leading strand and of each Okazaki fragments of lagging strand

Question 26

contribution of the following molecules in the process of replication: DNA ligase

Answer 26

Joins 3’ end of DNA that replaces primer to rest of leading strand and joins okazaki fragments of lagging strand.

Question 27

contribution of the following molecules in the process of replication: DNA polymerase I

Answer 27

Removes RNA nucleotides of primer from 5’ end and replaces them with DNA nucleotides

Question 28

contribution of the following molecules in the process of replication: DNA polymerase III

Answer 28

Using parental DNA as a template, synthesizes new DNA strand by covalently adding nucleotides to the 3’ end of a pre-existing DNA strand or RNA primer.

Question 29

contribution of the following molecules in the process of replication: Topoisomerase

Answer 29

breaking, swiveling, and rejoining the DNA strands to release the strain before the Helicase(ahead of replication fork)

Question 30

Understand that both _____ and _____ use energy in ATP hydrolysis for their role in DNA replication.

Answer 30

topoisomerase and helicase

Question 31

Replication always occurs in which direction? Why? What is required for DNA polymerase to make its first phosphodiester bond? What is primase’s role in meeting one of these needs?

Answer 31

Replication always occurs in 5’ to 3’ direction - this is because nucleotide triphosphates are only added on 3’ end where there is hydroxyl(-OH). What does DNA polymerase need to “do it’s job” - Anti-parallel template - dNTP’s (nucleotides) - Mg2+ (as a cofactor. First alpha phosphate form phosphodiester bond with OH- with Mg2+ as a catalyst. Another one Mg2+ bound to both beta and omega phosphate makes it easy to cleave them off. ) - 3’ OH RNA primer create short RNA primer complementary to template (5-10 nucleotides long)

Question 32

How does DNA polymerase know the correct nucleotide to add to the growing strand?

Answer 32

Base pairing (A-T, G-C)

Question 33

What is the primer made of? Understand the process of removing/replacing the primer during DNA replication. What enzymes are involved?

Answer 33

Primer is made of 5-10 RNA nucleotides. 1) Primase makes short RNA primers on the template strand 2) DNA polymerase III comes in and continues until it bumps into primer 3) DNA polymerase I comes and replace RNA ribonucleotide to DNA deoxyribonucleotides 4) Ligase seals the nick. Enzymes involved: primase. DNA polymerase III, DNA polymerase I, ligase

Question 34

17. Understand the following terms and be able to identify on a diagram or draw on a diagram: a. lagging strand b. leading strand c. Okazaki fragment

Answer 34

a. lagging strand synthesized discontinuously, in the opposite direction of replication fork. b. leading strand DNA synthesized continuously, in the direction of replication fork c. Okazaki fragment Made on lagging strand

Question 35

What is the relationship between the antiparallel nature of the double helix and the lagging and leading strands?

Answer 35

DNA polymerase can add nucleotides only in 5’ to 3’ direction. In leading strand it occurs continuously whereas in lagging strand, it needs multiple primers. Bidirectional replication. ( In a replication bubble, the top strand and the bottom strand each have a leading strand & lagging strand being replicated in opposite directions)

Question 36

What are the major differences between DNA replication in prokaryotes and eukaryotes?

Answer 36

- Multiple origins of replication for eukaryotes. - Prokaryotes have circular double stranded DNA; Eukaryotes have linear double stranded DNA. - Eukaryotic DNA contains many more base pairs - DNA polymerase III cannot add final sequence of DNA to the 5’ end of lagging daughter strand.

Question 37

Why do successive rounds of replication lead to shortening of the ends of linear DNA molecules in eukaryotes?

Answer 37

Because for the primer at the very end, there is no 3’ end to stick nucleotides to.

Question 38

How do eukaryotic cells postpone the erosion of genes near the ends of the chromosomes? What are the sequences at the ends of eukaryotic DNA molecules called? How do these sequences protect the information on chromosomes? What enzyme adds these sequences?

Answer 38

By adding telomeres(sacrificial nucleotides)by telomerase enzyme to ends of linear chromosomes. Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of the DNA molecules.

Question 39

What is the role of telomerase in the formation of gametes, cells of the embryo, stem cells, and cancer?

Answer 39

Telomerase is on. ( In human cells, most genes have telomerase off - it has been proposed that the shortening of telomeres is connected to aging). Telomerase prevents this in stem cells etc.

Question 40

Mutations need to be repaired before what stage of the cell cycle?

Answer 40

Before S phase

Question 41

23. What is meant when we say the DNA polymerases proofread newly made DNA? When does proofreading have to happen?

Answer 41

While DNA polymerase is replicating, it detects and repairs its mistakes – knows that it is wrong because the geometry is wrong. Proofreading has to occur during the replication.

Question 42

What is mismatch repair? How do the repair enzymes identify a “mismatch”? Explain how the mismatch repair system repairs mistakes in DNA. What role does DNA methylation play in this process? What enzymes are involved in this process? What is nucleotide excision?

Answer 42

Mismatch repair : - Incorrectly added base is detected after the replication & mismatch protein detect the base and remove it from newly synthesized strand. - (mismatch repair enzymes are scanning the newly produced DNA looking for distortions in the helix as a way to identify a mismatch because the geometry isn't right). They identify mismatch incorrect base in unmethylated strand and nick unmethylated strand - DNA polymerase I excises nucleotides on unmethylated strand - DNA polymerase I fills in the gap 5’ to 3’ direction - DNA ligase links new and old nucleotides. It involves mismatch repair enzymes, DNA polymerase I (cuts out abnormal base pairs, just like it removes RNA nucleotides) & ligase (seals the nick)