DNA Replication

Question 1

Southern Hybridization

Answer 1

Hybridization of a probe fragement to a DNA target

Question 2

Northern Hybridization

Answer 2

Hybridization of a probe fragement to a RNA target

Question 3

Requirements of PCR

Answer 3

Template (ss or ds)

Pair of primers

4 deoxyribonuclotides

Taq DNA polymerase

Appropriate buffer

Question 4

PCR: DNA is denatured at __ degrees and strand extension occurs at __ degrees

Answer 4

Denatured: 95 degrees celcius

Strand extension occurs at 72 degrees celcius which is the optimal temperature of Taq

Question 5

Equation for PCR

Answer 5

2^n where n = number of cycles

Question 6

How does microarray/chip based hybridization work?

Answer 6

Large numbers of gene-specific oligonuclotides printed/spotted on a chip are hybridized to total RNA and the number and intensity of hybridizing spots are determined

Question 7

What is the medical function of microarray/chip based hybridization

Answer 7

Allows us to address a treatment-specific question: Is there a difference in gene expression pattern between responders and non-responders of drugs

Question 8

Medically important applicaitons of DNA

Answer 8

Disease diagnosis

Prenatal diagnosis

Bacterial infections

Virus infection and viral load

Question 9

DNA matches are obtained by experimental analysis needs to be confirmed by

Answer 9

Statistical analysis

Question 10

Forensic medicine relies on

Answer 10

Variable number of tandem repeats (VNTR)

Question 11

Cell doctrine

Answer 11

Every cell in the body arises from an existing cell

Question 12

In what phase does DNA replication occur?

Answer 12

S phase

Question 13

2 main phases of cell cycle

Answer 13

Interphase + Mitosis

Question 14

Interphase consists of

Answer 14

G1

S

G2

Question 15

Direction DNA synthesized in

Answer 15

5’ to 3’

Question 16

5’ end of DNA contains

3’ end of DNA contains

Answer 16

5’: phosphate

3’: hydroxyl

Question 17

E. coli polymerase types

Answer 17

DNA Pol 1-3

Question 18

Processivity of DNA Pol I-III

Answer 18

DNA Pol I: low processivity

DNA Pol II: higher processivity

DNA Pol III: highest processivity

Question 19

Which DNA Pol do DNA repair

Answer 19

Pol I and II

Question 20

What is the function of DNA Pol III

Answer 20

DNA replication

5’ –> 3’ polymerization

3’ –> 5’ exonuclease

Question 21

What is the function of DNA Pol I

Answer 21

5’ –> 3’ polymerization

5’ –> 3’ exonuclease

3’ –> 5’ exonuclease

Question 22

dnaA =

Answer 22

initiator protein

Question 23

dnaB =

Answer 23

helicase

Question 24

dnaG =

Answer 24

primase

Question 25

In E. Coli replication, the initiator protein binds to the origin and denatures ___ base pairs

Answer 25

A-T

Question 26

dsDNA is unwound by

Answer 26

Helicase (dnaB) in an ATP-dependent manner

Question 27

Unwound strands of DNA are stabilized by

Answer 27

ss binding protein

Question 28

What relieves overwinding of of DNA as one part unwinds

Answer 28

Type II topoisomerase (gyrase)

Question 29

Primase synthesizes

Answer 29

15 nt long RNA primers

Question 30

What is the absolute requirement for RNA replication

Answer 30

Primers

Question 31

DNA polymerase requires ___ and __ for synthesis of a polynucleotide strand

Answer 31

Template Free 3' OH group

Question 32

DNA polymerase I- function

Answer 32

Removes RNA primers which are then replated by extending DNA synthesis

Question 33

E. Coli polymerase III is a __ enzyme

Answer 33

Holoenzyme

Question 34

What are the subunits of E. Coli Polymerase III (holoenzyme)

Answer 34

Catalytic core of 3 subunits (alpha-epsilon-theta) Clamp subunits (beta, beta) for processivity Clamp loader (gamma-complex) Assembly subunit (tau, tau)

Question 35

Logical target for inhibiting bacteria replicaiton

Answer 35

Enzymes of DNA replication

Question 36

Streptomyces spheroides and streptomyces niveus produces

Answer 36

Novobiocin

Question 37

What is novobiocin

Answer 37

Replicaiton/transcription inhibitor

Question 38

Apart from novobiocin, what are some other synthetic antibiotics

Answer 38

Fluoroquinolones = levofloxacin and ciprofloxacin

Question 39

Type I vs. Type II topoisomerase

Answer 39

Type I makes a cut on only 1 strand of DNA Type II = gyrase and makes 2 cuts (one on each strand of DNA)

Question 40

What is the target enzyme for fluoroquinoles?

Answer 40

Bacterial gyrase

Question 41

Gyrase is a ___ (structure)

Answer 41

Heterotetramer (GyrA2GyrB2)

Question 42

How does gyrase cleave DNA?

Answer 42

Tyr122 on GyrA forms covalent linkage with the 5'P of DNA GyrB carries out ATP hydrolysis

Question 43

How do fluoroquinolones inhibit Gyrase?

Answer 43

They prevent the revesible ligation step carried out by GyrA

Question 44

What competes with ATP for binding to GyrB

Answer 44

Novobiocin

Question 45

What is the target enzyme for anticancer drugs?

Answer 45

Mammalian Topo I

Question 46

Topoisomerase I binds to

Answer 46

3' end of phosphodiester back bone

Question 47

How does Topotecan work?

Answer 47

It stabilizes topoisomerase I-DNA complex and prevents the religation step = inhibition of replicaiton

Question 48

Resistance to quinolone may be caused by point mutations to

Answer 48

N-terminal end of GyrA (most mutations) C-terminal end of GyrB (few mutations)