DNA Replication Flashcards
Southern Hybridization
Hybridization of a probe fragement to a DNA target
Northern Hybridization
Hybridization of a probe fragement to a RNA target
Requirements of PCR
Template (ss or ds)
Pair of primers
4 deoxyribonuclotides
Taq DNA polymerase
Appropriate buffer
PCR: DNA is denatured at __ degrees and strand extension occurs at __ degrees
Denatured: 95 degrees celcius
Strand extension occurs at 72 degrees celcius which is the optimal temperature of Taq
Equation for PCR
2^n where n = number of cycles
How does microarray/chip based hybridization work?
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
What is the medical function of microarray/chip based hybridization
Allows us to address a treatment-specific question: Is there a difference in gene expression pattern between responders and non-responders of drugs
Medically important applicaitons of DNA
Disease diagnosis
Prenatal diagnosis
Bacterial infections
Virus infection and viral load
DNA matches are obtained by experimental analysis needs to be confirmed by
Statistical analysis
Forensic medicine relies on
Variable number of tandem repeats (VNTR)
Cell doctrine
Every cell in the body arises from an existing cell
In what phase does DNA replication occur?
S phase
2 main phases of cell cycle
Interphase + Mitosis
Interphase consists of
G1
S
G2
Direction DNA synthesized in
5’ to 3’
5’ end of DNA contains
3’ end of DNA contains
5’: phosphate
3’: hydroxyl
E. coli polymerase types
DNA Pol 1-3
Processivity of DNA Pol I-III
DNA Pol I: low processivity
DNA Pol II: higher processivity
DNA Pol III: highest processivity
Which DNA Pol do DNA repair
Pol I and II
What is the function of DNA Pol III
DNA replication
5’ –> 3’ polymerization
3’ –> 5’ exonuclease
What is the function of DNA Pol I
5’ –> 3’ polymerization
5’ –> 3’ exonuclease
3’ –> 5’ exonuclease
dnaA =
initiator protein
dnaB =
helicase
dnaG =
primase
In E. Coli replication, the initiator protein binds to the origin and denatures ___ base pairs
A-T
dsDNA is unwound by
Helicase (dnaB) in an ATP-dependent manner
Unwound strands of DNA are stabilized by
ss binding protein
What relieves overwinding of of DNA as one part unwinds
Type II topoisomerase (gyrase)
Primase synthesizes
15 nt long RNA primers
What is the absolute requirement for RNA replication
Primers
DNA polymerase requires ___ and __ for synthesis of a polynucleotide strand
Template
Free 3’ OH group
DNA polymerase I- function
Removes RNA primers which are then replated by extending DNA synthesis
E. Coli polymerase III is a __ enzyme
Holoenzyme
What are the subunits of E. Coli Polymerase III (holoenzyme)
Catalytic core of 3 subunits (alpha-epsilon-theta)
Clamp subunits (beta, beta) for processivity
Clamp loader (gamma-complex)
Assembly subunit (tau, tau)
Logical target for inhibiting bacteria replicaiton
Enzymes of DNA replication
Streptomyces spheroides and streptomyces niveus produces
Novobiocin
What is novobiocin
Replicaiton/transcription inhibitor
Apart from novobiocin, what are some other synthetic antibiotics
Fluoroquinolones = levofloxacin and ciprofloxacin
Type I vs. Type II topoisomerase
Type I makes a cut on only 1 strand of DNA
Type II = gyrase and makes 2 cuts (one on each strand of DNA)
What is the target enzyme for fluoroquinoles?
Bacterial gyrase
Gyrase is a ___ (structure)
Heterotetramer (GyrA2GyrB2)
How does gyrase cleave DNA?
Tyr122 on GyrA forms covalent linkage with the 5’P of DNA
GyrB carries out ATP hydrolysis
How do fluoroquinolones inhibit Gyrase?
They prevent the revesible ligation step carried out by GyrA
What competes with ATP for binding to GyrB
Novobiocin
What is the target enzyme for anticancer drugs?
Mammalian Topo I
Topoisomerase I binds to
3’ end of phosphodiester back bone
How does Topotecan work?
It stabilizes topoisomerase I-DNA complex and prevents the religation step = inhibition of replicaiton
Resistance to quinolone may be caused by point mutations to
N-terminal end of GyrA (most mutations)
C-terminal end of GyrB (few mutations)
