Module 8

Question 1

What is Chargaff’s rule?

Answer 1

the balance b/w A-T & G-C in ALL organisms

• DNA structure is REGULAR + STABLE

Question 2

What does X-ray crystallization tell us?

Answer 2

info abt the structure of DNA

• DNA is a double helix
• distance / angle between rings

Question 3

What are the uses of nucleotides?

Answer 3

energy storage + use

Question 4

Purines

Answer 4

A + G (2 rings)

Question 5

Pyrimidines

Answer 5

T + C + U (1 ring)

Question 6

RNA forms . . .

Answer 6

hairpins + loops

• can also have a hairpin double helix
• CAN have H-bonding by not consistent
• can form complex 3D structures

Question 7

Explain what it means that DNA is semi-conservative.

Answer 7

because when replication occurs, only one of the parent strains if retained; the 2nd strand is formed using new bases

Question 8

What is the sliding clamp?

Answer 8

assists DNA polymerase on both leading + lagging strand so that it can travel for a long time

makes replication more PROCESSIVE

Question 9

All polymerases are joined at the . . .

Answer 9

clamp loader

Question 10

What are the functions of DNA pol I?

Answer 10

• replaces RNA primers with DNA nucleotides on both strands
• involved in repair of DNA damage
• has 2 types of exonuclease activity

Question 11

What are the functions of DNA pol III?

Answer 11

the MAIN polymerase in replication

• adds nucleotides to template strand during replication
• 3’ –> 5’ exonuclease activity only

Question 12

Structure of DNA pol I

Answer 12

palm, fingers, thumb, insertion & post-insertion sites

only 1 subunit

Question 13

Structure of DNA pol III

Answer 13

over 10 subunits

Question 14

Function of DNA helicase

Answer 14

unwinds DNA helix; breaks H bonds

spinning motor of 6 subunits; hydrolyzes ATP + spins

Question 15

Function of SSB

Answer 15

bind to single strand, prevent them from rejoining

also protect the single strand from NUCLEASE attack

Question 16

Function of gyrase

Answer 16

binds ahead of helicase and reduces tension (relieves tensional strain created by DNA unwinding)

Question 17

Function of primase

Answer 17

adds RNA primer

Question 18

Function of ligase

Answer 18

seals nicks in lagging strand

Question 19

DnaA

Answer 19

recognizes Ori sequence; opens duplex at specific sites in origin

Question 20

What is the origin of replication?

Answer 20

sequence at which replication begins

Question 21

Describe the initiation of DNA replication at the origin of replication

Answer 21

• replication bubble forms - at either end of this bubble is a REPLICATION FORK
• eventually, the forks move around & join up so that the entire chromosome is replicated
• more origins of replication are found in eukaryotes

Question 22

Explain DNA replication on the lagging strand

Answer 22

1) RNA primer added to fragment 2

2) DNA pol III adds nucleotides in 5’ –> 3’ direction until it reaches fragment 1

3) sliding clamp runs loose, & a new one is added

4) DNA pol I replaces the RNA w/ DNA, adding to the 3’ end of fragment 2

5) DNA ligase forms a bond b/w the newest DNA & the end of fragment 1

Question 23

DNA synthesis works by adding nucleotides at the 3’ end of the. . .

Answer 23

NEW strand

Question 24

What is a telomere?

Answer 24

a sequence at the end of chromosomes (that protect the ends) on the lagging strand. The sequence at the very end cannot be replicated due to the absence of a 3’ end for DNA polymerase

Telomerase is an enzyme that adds telomeres to the ends & this prevents the shortening of the chromosomes

Question 25

How is DNA fidelity maintained?

Answer 25

1) proof-reading ability

2) complementary bases (binding of nucleotides)

Question 26

What are the 2 main active sites of DNA polymerase?

Answer 26

1) polymerization (adding nucleotides)

2) exonuclease activity (removing them)

Question 27

What is a vector?

Answer 27

something that will hold a piece of DNA

Question 28

What are resistance sites on a plasmid vector?

Answer 28

sequence(s) that allow us to select ONLY the bacteria that holds this plasmid & ALL of the other bacteria will die

Question 29

What are restriction sites?

Answer 29

Some sequences where we can insert the DNA that we want

Question 30

What are restriction enzymes?

Answer 30

1) recognize short PALINDROME sequences

2) cut a specific sequence

3) some generate sticky ends, others blunt ends

4) compatible ends can base-pair (& then joined by DNA ligase) to form recombinant DNA

Question 31

What are the important features of E coli plasmid vectors for use in cloning DNA?

Answer 31

• origin of replication
• resistance sites
• restriction sites

Question 32

restriction enzymes are actually. . .

Answer 32

VIRAL defense proteins from bacteria

Question 33

What sections of a vector are needed for the expression of a protein?

Answer 33

1) bacterial promoter (P) or operator (O) sequences

2) poly-liker / multi-cloning sites (an area w many restriction enzyme sites)

3) selectable genetic marker (e.g. antibiotic resistance)

4) ori

5) gene encoding repressor that binds O and regulates P

Question 34

You can express a protein for. . .

Answer 34

a) functional assays
b) in a model organism
c) in human cells in cell culture

Question 35

Why are DNA primers used in PCR?

Answer 35

• more stable
• much easier to use in lab
• bc RNA degrades very easily
• we can buy them on the internet