Biochem - Molecular (DNA Replication, Mutations, & Repair)

Question 1

Compare/Contrast DNA replication in eukaryotes and prokaryotes.

Answer 1

Eukaryotic DNA replication is more complex than the prokaryotic process but uses many enzymes analogous to those listed below. In both prokaryotes and eukaryotes, DNA replication is semiconservative and involves both continuous and discontinuous (Okazaki fragment) synthesis.

Question 2

What is the origin of replication, and what purpose does it serve? Is it single or are their multiple origins?

Answer 2

Particular consensus sequence of base pairs in genome where DNA replication begins. May be single (prokaryotes) or multiple (eukaryotes).

Question 3

What is the Replication fork, and what purpose does it serve?

Answer 3

Y-shaped region along DNA template where leading and lagging strands are synthesized.

Question 4

What is the function of Helicase?

Answer 4

Unwinds DNA template at replication fork

Question 5

What is the function of Single-stranded binding proteins?

Answer 5

Prevent strands from reannealing

Question 6

What is the function of DNA topoisomerases?

Answer 6

Create a single- or double-stranded break in the helix to add or remove supercoils

Question 7

What is the mechanism of Fluoroquinolones?

Answer 7

Fluoroquinolones - inhibit DNA gyrase (prokaryotic topoisomerase II)

Question 8

What is the function of Primase?

Answer 8

Makes an RNA primer on which DNA polymerase III can initiate replication

Question 9

In what organisms is DNA polymerase I versus III found?

Answer 9

DNA polymerase I (Prokaryotic only) & DNA polymerase III (Prokaryotic only)

Question 10

What is the function of DNA polymerase III?

Answer 10

Elongates leading strand by adding deoxynucleotides to the 3’ end. Elongates lagging strand until it reaches primar of preceding fragment. 3’ => 5’ exonuclease activity “proofreads” each added nucleotide. DNA polymerase III has 5’ => 3’ synthesis and proofreads with 3’ => 5’ exonuclease.

Question 11

What is the function of DNA polymerase I? How does it compare/contrast to DNA polymerase III?

Answer 11

Degrades RNA primer; replaces it with DNA; Has same functions as DNA polymerase III but also excises RNA primer with 5’ => 3’ exonuclease.

Question 12

What is the function of DNA ligase?

Answer 12

Catalyzes the formation of a phosphodiester bond within a strand of double-stranded DNA (i.e., joins Okazaki fragment). Seals.

Question 13

What is Telomerase, and what function does it have?

Answer 13

An RNA-dependent DNA polymerase that adds DNA to 3’ ends of chromosomes to avoid loss of genetic material with every duplication.

Question 14

Draw a zoomed in version of DNA replication, including and labeling the following: (1) Fork movement (2) Leading strand (3) Lagging strand (4) Area of interest (5) Origin of replication.

Answer 14

See p. 69 in First Aid 2014 for visual at bottom left of page

Question 15

Draw DNA replication, including and labeling the following: (A) Origin of replication (B) Replication fork (C) Helicase (D) Single-stranded binding protein (E) Topoisomerase (F) Primase (G) DNA polymerase III (H) DNA polymerase I (I) DNA ligase.

Answer 15

See p. 69 in First Aid 2014 for visual at bottom right

Question 16

List the 4 major types of DNA mutations in order of increasing severity.

Answer 16

Severity of damage: silent &laquo_space;missense < nonsense < frameshift

Question 17

What is a transition versus transversion? To what kinds of mutations do they apply?

Answer 17

For silent, missense, and nonsense mutations: TRANSITION - purine to purine (e.g., A to G) or pyrimidine to pyrimidine (e.g., C to T), TRANSVERSION - purine to pyrimidine (e.g., A to T) or pyrimidine to purine (e.g., C to G)

Question 18

What defines a silent DNA mutation? Where does it often occur, and why?

Answer 18

Nucleotide substitution but codes for same (synonymous) amino acid; often base change in 3rd position of codon (tRNA wobble)

Question 19

What defines a missense DNA mutation? In what context is it considered conservative?

Answer 19

Nucleotide substance resulting in changed amino acid (called conservative if new amino acid is similar in chemical structure)

Question 20

What is a clinical example of a missense DNA mutation?

Answer 20

Sickle cell disease

Question 21

What defines a nonsense DNA mutation?

Answer 21

Nucleotide substitution resulting in early stop codon; Think: “STOP the NONSENSE!”

Question 22

What defines a frameshift DNA mutation? What is usually the result of this? What is a clinical example of this?

Answer 22

Deletion or insertion of a number of nucleotides not divisible by 3, resulting in misreading of all nucleotides downstream, usually resulting in a truncated, nonfunctional protein; Duchenne muscular dystrophy

Question 23

Describe the process of Nucleotide excision repair. For what general purpose is it used?

Answer 23

Specific endonucleases release the oligonucleotide-containing damaged bases; DNA polymerase and ligase fill and reseal the gap, respectively; Repairs bulky helix-distorting lesions.

Question 24

In what condition is Nucleotide excision repair defective? What characterizes this condition?

Answer 24

Defective in Xeroderma pigmentosum, which prevents repair of pyrimidine dimers because of ultraviolet light exposure.

Question 25

Describe the process of Base excision repair, including key factors and their functions.

Answer 25

Base-specific glycosylase recognizes altered base and creates AP site (apurinic/apyrimidinic). One or more nucleotides are removed by AP-endonuclease, which cleaves the 5’ end. Lyase cleaves the 3’ end. DNA polymerase-Beta fills the gap and DNA ligase seals it.

Question 26

In what context is Base excision repair important?

Answer 26

Important in repair of spontaneous/toxic deamination

Question 27

Briefly describe the process of Mismatch repair.

Answer 27

Newly synthesized strand is recognized, mismatched nucleotides are removed, and the gap is filled and resealed.

Question 28

In what condition is Mismatch repair defective?

Answer 28

Defective in hereditary nonpolyposis colorectal cancer (HNPCC)

Question 29

What are 3 types of single strand DNA repair?

Answer 29

(1) Nucleotide excision repair (2) Base excision repair (3) Mismatch repair

Question 30

What is a type of double strand DNA repair?

Answer 30

Nonhomologous end joining

Question 31

What is the purpose of Nonhomologous end joining? Is homology required?

Answer 31

Brings together 2 ends of DNA fragments to repair double-stranded breaks. No requirement for homology.

Question 32

In what condition is Nonhomologus end joining mutated?

Answer 32

Mutated in ataxia telangiectasia