DNA Replication and Repair

Question 1

What is semiconservative replication?

Answer 1

the use of each strand as a template for synthesis

Question 1

What is base pairing?

Answer 1

the recognition of the parental nucleotide and the incorporation of a complementary base in the newly synthesized strand

Question 2

What is DNA polymerase I and what are the requirements for its function? What is the alternative name?

Answer 2

Catalyzed the polymerization of nucleotides. It requires: all four deoxyribonucleotides (dATP, dGTP, dCTP, dTTP), Mg, a 3’ OH primer and a template

• The function of the primer is to start the polymerization, then it will be replaced by polymerase I
• Polymerase I is also called Kornberg enzyme. It synthesizes in 5’ to 3’ direction and has exonuclease activity (providing both repair and proofreading)

Question 3

In which direction does proof reading progress?

Answer 3

3’ to 5’
* The wrong nucleotide can not hydrogen bond to the opposing template and causes polymerization to stop until the error is repaired

Question 4

In which direction does the repair progress?

Answer 4

5’ to 3’

Question 5

What is the function of polymerase II , III?

Answer 5

polymerase II function is unknown

polymerase III function is similar to polymerase I

Question 6

What is DNA ligase?

Answer 6

Forms phosphodiester bonds between 3’ OH and 5’ mono phosphate groups. This is an endergonic reaction requiring energy by NAD (bacteria) or ATP (viruses and animals)

Question 7

What is primase?

Answer 7

An enzyme that synthesizes a short RNA strand (4-10 bases) complementary to the template (a primer), required by polymerase for DNA synthesis. Its growing end is 3’ OH

Question 8

What is the helicase enzyme?

Answer 8

unwind the double DNA helix before replication can occur. It requires ATP

Question 9

What is the Single strand binding protein SSB?

Answer 9

It prevents the re-annealing of the DNA strand and also protects it from the cleavage of nucleases

Question 10

What is the functions of topoisomerase enzymes?

Answer 10

create swivel points in the DNA to relieve the strain created by the replication fork by cutting and resealing the DNA

Question 11

What is the function of the two topoisomerase enzyme?

Answer 11

1. Topoisomerase I: relaxes the DNA in front of the replication fork by creating a transient nick in one strand. Doesn’t require ATP
2. Topoisomerase II: introduces negative supercoils in the molecule by cutting and resealing the two strands. Requires ATP.
   * Gyrase is similar to topoisomerase II in E.Coli (introduces negative supercoils)
   see p. 378

Question 12

What is the difference between the leading strand and Okazaki fragment in DNA synthesis?

Answer 12

1. The leading strand: the one continuously synthesized in 5’-3’ direction (complementary to parental 3’ - 5’)
2. The lagging strand: this one is synthesized in short fragments which are joined later by ligase

Question 13

The replication fork is …..

Answer 13

the site where the two strands are unwound. It can move in either direction from the origin of replication (called ori)

Question 14

The separation of the strands is maintained by …..

Answer 14

binding of several Single strand binding proteins

Question 15

The synthesis of each of the Okazaki strands require …….

Answer 15

a short RNA primer

Question 16

Two replication forks forms at each …. site and move in an opposite direction

Answer 16

ori

Question 17

E Coli has ….. site, while the eukaryotic DNA has many

Answer 17

one ori

Question 18

The RNA primer (for DNA synthesis) is synthesized by a complex containing …… & …..

Answer 18

primase & single strand binding protein

Question 19

The primer on the lagging strand is located near ……

Answer 19

the replicating fork

Question 20

The free 3’-OH on the primer serve as the binding site for …..

Answer 20

the first deoxyribonucleotide added by the polymerase

Question 21

In the lagging strand, replication continues until ……

Answer 21

it reaches another RNA primer

Question 22

What happens when polymerase III reach another primer in Okazaki fragment?

Answer 22

It dissociates, allowing polymerase I to enter and erase the ribonucleotides one at a time from the 5’ end, then add deoxyribonucleotides to fill the gap
Ligase then forms the phosphodiester bonds between the fragments

Question 23

DNA replication is inhibited by analogs of ….., …. & ….

Answer 23

pyrimidines, purines & folate

* used to slow division of cancer cells

Question 24

Thymidylate synthase is an enzyme critical for ......., inhibited by .....

Answer 24

DNA synthesis | Pyrimidine analogs

Question 25

Cytosine arabinoside is an analog of the pyrimidine ......, used to ......

Answer 25

cytosine nucleoside stop elongation of the DNA chain * It lacks 3'-OH

Question 26

Aminopterin and methotrexate are .....

Answer 26

inhibitors of DHF acid reductase | see p. 380

Question 27

Define Mutation and Mutagen

Answer 27

* Mutation: is heritable change in DNA | * Mutagen is a chemical that alter the nucleotides in DNA

Question 28

Point mutation is a ....., and it is of two types, ..... & .....

Answer 28

change in a single nucleotide | transition, transversion

Question 29

What is transition mutation?

Answer 29

Transition: is the change of one purine or pyrimidine with another purine or pyrimidine respectively, due to mispairing of bases during replication or chemical insult.

Question 30

What is transversion mutation?

Answer 30

The exchange of a purine to pyrimidine or vice verse

Question 31

The nucleotide excision-repair mechanism repairs .....

Answer 31

UV damage

Question 32

Dimers are formed between ........ due to UV light exposure. It prevents ......

Answer 32

``` adjacent pyrimidines (usually thymidines) normal gene expression and replication ```

Question 33

Explain the nucleotide excision-repair theory

Answer 33

1. incision: by endonuclease in the phosphodiester bonds several nucleotides away from the dimer 2. Removal: by helicase which removes the hydrogen bonds 3. Polymerization: by polymerase I, which fills the gap in 5' - 3' direction 4. Ligation: by ligase

Question 34

Explain the base excision repair theory

Answer 34

Repairs most common error of deamination of cytosine to uracil (transition) causing U-G instead of C-G 1. Removal of Uracil base: by glycosidase 2. Cleavage of the phosphodiester bond next to missing base by endonuclease (belong to AP nucleases family) 3. Insertion of cytosine and ligation

Question 35

The enzymes topoisomerase II, ligase & helicase require .....

Answer 35

ATP to function

Question 36

What is Missense mutation??

Answer 36

A codon with an altered amino acids sequence | * example is valine replaces glutamate causing sickle cell anemia

Question 37

What is a nonsense mutation??

Answer 37

A stop codon that causes polypeptide chain termination

Question 38

What is the wobble effect??

Answer 38

in translation, only the first two base pairs of the codon is translated into anticodon by tRNA, ignoring the last (3rd) base pair

Question 39

What is the frameshift mutation??

Answer 39

the deletion of insertion of one or more base pairs in the template

Question 40

What is the function of the enzyme reverse transcriptase?

Answer 40

formation of a complementary DNA strand from RNA template.. * HIV has this enzyme * AZT is a reverse transcriptase inhibitor

Question 41

Polymerase chain reaction is used in .....

Answer 41

forensic medicine | * The lab sequence is : splitting the DNA strands, annealing primers to each one, copying each strand with polymerase