Kandpal 1 Flashcards
DNA sequence is important for…
- designing treatment options
- rational basis for treatment outcomes
- understanding disease mechanisms
___ and ___ genomes have been sequenced completely
human and chimpanzee
differences in human and chimp genomes
35 million single nucleotide changles
5 million insertion/deletion changes
various rearrangements
___% difference between human and chimp DNA sequence
4%
Differential susceptibilities to ___between human and chimps
malaria
Cytochrome P450 (CYP)
enzymes carry out several reactions including hydroxylation (activation/inactivation) of drugs.
___ may alter the activity of CYP enzymes
Polymorphisms
Drug dose needs to be adjusted based on genotype
Warfarin, a blood thinner, dose is dependent on the genotype (isoforms of CYP enzymes) of patients
CRISPR/Cas technology
Clustered Regularly Interspaced Short Palindromic Repeats
(Specific mutations in DNA sequence can be corrected by using genome editing tools)
CRISPR exist in bacteria along with helicase and nuclease
DNA bases
adenine guanine cytosine thymine (RNA has uracil instead of thymine)
5 carbon sugar in DNA/RNA
deoxyribose in DNA
ribose in RNA
nucleotides are joined by ___ ___
phosphodiester bonds.
5’ phosphate bonds to 3’ OH of next nucleotide
sugar-phosphate forms the
DNA backbone
2 strands of DNA of held by
H-bonds
purines
adenine and guanine
pyrimidines
cytosine, thymine,uracil
adenine deamination –>
hypoxanthine
guanine deamination –>
xanthine
uracil methylated –>
thymine
nucleoside
base + sugar
end in -ine
nucleotide
nucleoside (base + sugar) + phosphate
mono- di- or tri- phosphate.
end in -ylate
Nucleotide functions
part of DNA/RNA
cofactors
energy (ATP)
cell signaling (GTP in G protein coupled receptor)
FAD, NAD use
electron carriers
cyclic AMP and cGMP use
secondary messengers
coenzyme A (CoA) use
carbon carrier in fatty acid metabolism
Gregor mendel history
1866
unit factors/genetic determinants (genes)
debate on genetic material
DNA or proteins?
Griffith’s experiment.
used?
1928
DNA is genetic material
used Streptococcus pneumoniae
Virulent strain- S (smooth polysaccharide coat)
Avirulent strain- R (rough)
_________ purified the infection causing chemical as DNA.
Avery, McCarty and MacLeod
Hershey and Chase experiment
Which component of a bacteriophage enters the bacteria during infection?
Chargaff’s rule
The abundance of A equals T, and C equals G in double stranded DNA (not in a single stranded DNA)
A=T and C=G does not hold good for single stranded DNA
Watson and Crick model
double helix structure held together by H bonds.
Biophysical data from X-ray diffraction /Crystal structure
Double Helix explained ___ synthesis of DNA
templated
H-bonded base-pairs in DNA can be ___and ___
denatured and renatured
denaturation follows
zero order kinetics
at specific temp, does not depend on concentration of DnA
renaturation follows
second order kinetics
dependent on concentration of both strands
A DNA
right handed, 11 bases/turn
B DNA
right handed, 10 bases/turn
Z DNA
left handed, 12 bases/turn
we primarily see ___ DNA in living cells
B DNA
hyperchromicity curves
Double stranded: absorbance doubles when 2 strands fall apart. almost 100% increase.
Single stranded: increases b/c strand gets straighter as H bonds are denatured within strand. only 10-15% increase.
heat
disrupts H bond
increase in [Na+] [K+]
stabilizes helix
increase in GC/AT ratio
stabilizes helix
GC is 3 bonds
AT is 2 bonds
urea and formamide
destabilize DNA helix
hybridization
annealing of complementary DNA strands
PCR
polymerase chain reaction
primer-template annealing
southern hybridization
hybridization of a probe fragment to DNA target
northern hybridization
hybridization of a probe fragment to RNA target
microarrays
- global profiling of gene expression
- nucleotide polymorphisms
medically important applications of hybridization
- Disease diagnosis, Prenatal diagnosis, bacterial infections, virus infection and viral load
- Forensic medicine (Paternity test; Guilty vs Innocent verdict)
- -genotypic variations among individuals
___ are different in prokaryotic and eukaryotic DNA replication
Identities of proteins involved are different
Medical applications of molecular differences in prokaryotic and eukaryotic DNA replication
Differences in molecular machinery has allowed development of therapeutics (antibiotics)
cell doctrine
Every cell in the body arises from an existing cell.
cell cycle
orderly sequence of DNA duplication and cell division
DNA Replication is ___
Semiconservative
new DNA molecule has one parent strand and one new strand.
DNA Polymer Extends in ___ Direction
5’ to 3’
requires a primer in antiparallel orientation.
E. coli polymerase types
DNA Polymerase I
- Low processivity (DNA repair)
DNA Polymerase II
- Somewhat higher processivity (DNA repair)
DNA Polymerase III (DNA Replicase)
-Highest processivity (DNA replication)
DNA replicase
DNA polymerase III
Pol I activity
5’-3’ polymerization
5’-3’ exonuclease
3’-5’ exonuclease
Pol II activity
5’-3’ polymerization
3’-5’ exonuclease
Pol II (replicase) activity
5’-3’ polymerization
3’-5’ exonuclease
The primer attaches to the ___ end of the template strand.
3’
proofreading activity is …
3 ‘to 5’ exonuclease activity.
Pol III
adult humans have how many cells
10^14
% of human DNA is coding DNA
1%
E. coli error frequency after proofreading.
10^-9
human genome error rate
3/cell/replication
genome type E. coli
double stranded circular genome
replication beings at
origin of replication
__ binds to the origin or replication and denatures ___ base pairs
initiator protein (dnaA) A-T
double stranded DNA is unwound by
helicase (dnaB)
in an ATP dependent manner
strand unwound by helicase is stabilized by…
single-strand binding protein
Unwinding of one part of DNA leads to ___of other parts of DNA
overwinding
___ relieves overwinding
Type II topoisomerase (gyrase in E. coli)
makes a cut in DNA
___ synthesizes ___ nt long ___ primers
primase (dnaG)
15 nt long
RNA
___ are absolute requirements for replication
primers
DNA polymerase requires a ___ and a ___ with ___ group for the synthesis of a polynucleotide strand
template
primer with free 3’-OH group
___ adds new nucleotides to the 3’-OH end of the growing chain by copying the template sequence
DNA polymerase III (replicase, dnaE)
___ removes RNA primers
DNA polymerase I ( 5 to 3 exonuclease activity)
RNA primers are replaced by
extending DNA syntehesis
___ seals the gap in ____ ___ manner
DNA ligase
in ATP dependent manner
Polymer II is a ___
holoenzyme
holoenzyme subunits
- Catalytic core of three subunits a-e-q,
- Clamp subunits (b, b) for processivity
- Clamp loader (g complex)
- Assembly subunit (t, t)
Differences in mammalian and bacterial proteins can be exploited for ___
therapeutics:
Selective targeting of infectious agents by antibiotics
targets for bacteria elimination
Enzymes of DNA replication, transcription, translation, cell wall synthesis
Microbes use ___ for killing neighboring microbes
natural products (metabolites)
Novobiocin is ___ inhibitor
replication
fluoroquinolones
inhibitors of?
examples
synthetic antibiotics
inhibitors of replication
levofloxacin, ciprofloxacin (FDA approved for anthrax infection)
Streptomyces niveus produce an ___class of antibiotics such as ___(replication inhibitor
aminocoumarin
Novobiocin
___make transient cuts on DNA and change linking number (Total # of basepairs/10.4)
Topoisomerases
type I topoisomerases
make a nick in only one strand of DNA and binds to 3’ end of phosphodiester back bone
type II topoisomerases
eg. bacterial DNA gyrase
make two cuts, one in each strand.
___ and ___ are target enzymes for quinolones/fluoroquinolones
Bacterial DNA gyrase (Topoisomerase type II) and topoisomerase IV
gyrase is ___ (A2B2)
heterotetramer
- To make the cut Tyr122 on GyrA forms covalent linkage with the 5’P of DNA. The DNA is subsequently religated.
- GyrB carries out ATP hydrolysis.
topoisomerase IV is …
heterotetramer (C2E2)
fluoroquinolones prevent ….
the reversible ligation step carried out by GyrA
Novobiocin competes with ___ for binding to ___
ATP
GyrB
___ is the target enzyme for anticancer drugs
mammalian topo I
Topotecan (Hycamtin) is FDA approved for ___
ovarian cancer and small cell lung cancer.
Topotecan stabilizes Topo I-DNA complex and prevents the religation step (inhibition of replication)
logic for antibiotic resistance
- Bacterial genomes are small
- Replication and doubling times are shorter
- Errors in replication are likely to occur
- These errors lead to mutations and produce mutant bacteria
resistance to quinolone, point mutations
N-terminal end of GyrA (most mutations).
C-terminal end of GyrB (few mutations).
more proteins in ___ replication system
eucaryotic
genome size E. coli vs mammalian
E. coli - 4.64 million basepairs, circular DNA
mammalian - 23 pairs of linear chromosomes, 3.2 billion basepaires
___ have single origin of replication
E. coli
Mammalian uses ___ origins or replication
multiple (about 1000)
sequence not well defined
E. coli uses the ___ polymerase for leading and lagging strand synthesis
same
mammalian uses ____
polymerases for leading and lagging strand synthesis
Mammalian RNA primer is degraded by
RNaseH
RNaseH degrades RNA part of an RNA-DNA hybrid
Mammalian Okazaki fragments-
0.1- 0.2 Kb
E. coli Okazaki fragments-
1.0- 2.0 Kb
Polymerase alpha
primase
Polymerase beta
repair
Polymerase gamma
mitochondrial replication
Polymerase delta
nuclear replication
Polymerase epsilon
nuclear replication
end replication problem
In linear mammalian chromosomes, when replication fork reaches the end, RNA primer can’t be placed for the last Okazaki fragment
- Once the RNA primer is removed from the 5’ end, it can not be filled up.
- No polymerase available with 3’-5’ activity
- 5’ ends will shorten after each replication cycle
telomeres
maintain integrity of chromosome
seal end of chromosome
- Prevent undesirable fusion
- Prevent aberrant recombination
- Telomeres attach chromosomes to nuclear envelope
- Telomeres facilitate replication
telomeres have ___ repeats
hexameric
TTAGGG in human cells (up to 15 Kb)
TTGGGG in Tetrahymena
telomerase
Ribonucleoprotein (RNP)
Protein + RNA
synthesize telomeric repeats
reverse transcriptase (RNA dependent DNA polymerase)
telomerase activity
Somatic cells (differentiated cells) have no detectable activity
Germ cells, stem cells have detectable activity
telomerase and human cancer
normal tissue - no telomerase
tumor - activity
germ cells - activity
cancer, targets for intervention
-Telomerase activity
Inhibition would prevent tumor growth
-Telomere structure
Interference may prevent telomere extension
