DNA Structure, Analysis, Replication and Repair Flashcards
Characteristic of Genetic Material
- replication
- storage of information
- expression of information
- variation by mutation
Brief History in DNA discovery as the carrier of genetic material
- Friedrich Miescher - isolated cell nuclei and derived an acidic substance, now known to contain DNA, that he called nuclein
- Phoebus A. Levene - DNA contained approximately equal amounts of four similar molecules called nucleotides
- Erwin Chargaff - disproved Levene’s as incorrect; demonstrated most organisms do not contain precisely equal proportions of the four nucleotides
- Frederick Griffith - experiments with several different strains of the bacterium Diplococcus pneumoniae; transformation
- Avery, MacLeod, McCarty - first direct experimental proof that DNA, and not protein, is the biomolecule responsible for heredity; (protease, RNAse, and DNAse)
(6. Harvey & Chase) - T2 bacteriophage
Alternative models of DNA replication
- Semi-conservative model
- Conservative model
- Dispersive model
- at each point along the chromosome where replication is occurring, the strands of the helix are unwound
- if replication is bidirectional, two such forks will be present, migrating in opposite directions away from the origin
Replication fork
- refers to the length of DNA that is replicated following one initiation event
Replicon
Leading strand: Synthesized continuously in the same direction as the replication fork.
Lagging strand: Synthesized in small fragments called Okazaki fragments in the opposite direction of the replication fork.
four essential components required for DNA synthesis:
- dNTPs: dATP, dTTP, dGTP, dCTP (deoxyribonucleoside 5’-triphosphates)(sugar-base + 3 phosphates)
- DNA template
- DNA polymerase (Kornberg enzyme)
- Mg 2+ (optimizes DNA polymerase activity)
ability to remove nucleotides from the 3’ end of the chain
3’ to 5’ exonuclease activity
Five common DNA polymerases from mammals:
Polymerase (alpha):
* nuclear, DNA replication, no proofreading
Polymerase (beta):
* nuclear, DNA repair, no proofreading
Polymerase (gamma):
* mitochondria, DNA repl., proofreading
Polymerase (delta):
* nuclear, DNA replication, proofreading
Polymerase (epsilon):
* nuclear, DNA repair (?), proofreading
Segments of single-stranded DNA are called
_________
template strands
_______ (a type of topoisomerase) relaxes the supercoiled DNA; relieves torsional stress
Gyrase
_______ and _______ binds to the DNA at the replication fork and untwist the DNA using energy derived from ATP (adenosine triphosphate).
Initiator proteins
DNA helicase
DNA primase next binds to helicase producing a complex called a ______(primase is required for synthesis)
primosome
Primase synthesizes a short RNA primer of 10-12 nucleotides, to which DNA polymerase III adds nucleotides.
The RNA primer is removed and replaced with DNA by polymerase I, and the gap is sealed with ______
DNA ligase
_______ stabilize the single-stranded template DNA during the repliction process.
Single-stranded DNA-binding (SSB) proteins (>200)
Model of Replication (in E. coli)
- Initiator proteins bind to replication origin
- DNA helicase binds to replicator proteins
- Helicase loads onto DNA
- Helicase denatures helix and binds with DNA primase to form primosome
- Primase synthesizes RNA primer, which is extended as DNA chain by DNA Polymerase
an enzyme that joins DNA strands together by forming a phosphodiester bond between adjacent nucleotides; does not add a nucleotide
ligase
Each eukaryotic chromosome is one linear DNA double helix
Average ~10^8 base pairs long
With a replication rate of 2 kb/minute, replicating one human chromosome would require ~35 days.
Solution —> DNA replication initiates at many different sites simultaneously
binds to the terminal telomere repeat and catalyzes the addition of new repeats.
Telomerase
Most DNA repair occurs in the ___ phase of the eukaryotic cell cycle
G1 phase
Mismatch repair occurs in the ___ phase to correct replication errors
G2
encodes a protein that prevents a cell with damaged DNA from entering the S phase
p53 gene
a rare, inherited disorder that increases the risk of developing many types of cancer, especially at a young age
Li-Fraumeni syndrome
Maxam-Gilbert Method
Sanger Method
Meselson-Stahl experiment = ^15N-containing medium in ^14N medium
Taylor-Woods-Hughes Experiment - labeled chromatids
only a single region, called _____ , where replication is initiated
oriC
may be achieved on both the leading and lagging strands at a single replication fork
lagging template strand is “looped” in order to invert the physical direction of synthesis, but not the biochemical direction
enzyme functions as a dimer, with each core enzyme achieving synthesis on one or the other strand
Concurrent DNA Synthesis
DNA ligase seals the gaps between Okazaki fragments with a phosphodiester bond
a ligase seals the loose ends, creating hybrid duplexes called ______, held together by a cross-bridge structure [recombination process]
heteroduplex DNA molecules
position of this cross bridge can then move down the chromosome by a process referred to as ______
branch migration
Process of recombination:
a. two paired DNA duplexes, or homologs
b. endonuclease nicking
c. strand displacement
d. ligation, creating heteroduplex DNA molecules
e. branch migration
f. duplex separation
g. 180 degree rotation; x (chi) form or Holliday structure
h & i. if the two strands on opposite homologs are now nicked by an endonuclease ligation occurs; two recombinant duplexes are created
How PCR Works
Initial melt - 2 mins - 94C
Melt - 30 secs- 94C
Anneal - 30 secs - 55C
Extend - 1 min - 72C
Final extenson - 6 mins - 72C
Hold - 4C
PCR processe (ONE Cycle)
- Denaturing (95C)
- Annealing (55C)
- Extension (72C)
Three main features of the DNA
synthesis reaction:
- DNA polymerase I catalyzes formation of phosphodiester bond between 3’-OH of the deoxyribose (on the last nucleotide) and the 5’-phosphate of the dNTP.
• Energy for this reaction is derived from the release of two of the three phosphates of the dNTP. - DNA polymerase “finds” the correct complementary dNTP at each step in the lengthening process.
• rate ≤ 800 dNTPs/second
• low error rate - Direction of synthesis is 5’ to 3’
•Without proofreading error rate (mutation rate) is _______
•With proofreading error rate is _____ (1000-fold decrease)
1 x 10^-6
1 x 10^-9
DNA replication is continuous on the
______ strand and semidiscontinuous on
the _____ strand
leading
lagging
DNA replication in eukaryotes:
* Copying each eukaryotic chromosome during the S phase of the cell cycle presents some challenges:
Major checkpoints in the system:
- Cells must be large enough, and the environment favorable.
- Cell will not enter the mitotic phase unless all the DNA has replicated.
- Chromosomes also must be attached to the mitotic spindle for mitosis to complete.
- Checkpoints in the system include proteins call cyclins and enzymes called cyclin-dependent kinases (Cdks).
Enzymes that synthesize nucleic acids by
forming phosphodiester(PDE) bonds
Polymerases
Enzymes that hydrolyze PDE bonds
Nucleases
removes nucleotides from either the5’
or the 3’ end of a nucleic acid.
Exonucleases
cut within the nucleic acid and release
nucleic acid fragment
Endonucleases
substrates for DNA synthesis are
the dNTPs, whereas the substrates
for RNA synthesis are the NTPs.
STEPS OF DNA REPLICATION
- The base sequence at the origin of replication is recognized and bound by the dnaA protein.
- The two parental strands of DNA are pulled apart to form a “replication bubble “
- Helicase uses energy from ATP to break the hydrogen bonds holding the base pairs together
- Single-stranded DNA binding protein (SSB) binds to the single-stranded portion of each DNA strand, preventing the strands from reassociating and protecting them from degradation bynucleases.
- Primase synthesizes a short (about 10 nucleotides) RNA primer in the 5’ ~3’ direction, beginning at the origin on each parental strand.
- DNA polymerase III begins synthesizing DNA in the 5’ ~3’ direction, beginning at the 3‘ end of each RNA primer.
- RNA primers are removed by DNA polymerase I.
- Both DNA polymerase I and III have the ability to “proofread” their work by means of a 3’ ~5’ exonuclease activity.
8.DNA ligase seals the “nicks” between Okazaki fragments, converting them to a continuous strand of DNA. - DNA gyrase (DNA topoisomerase II) provides a “swivel” in front of each replication fork.
✓ DNA gyrase inserts negative supercoils by nicking both strands of DNA, passing the DNA strands through the nick, and then resealing both strands again.
can relieve supercoiling in DNA molecules by the transient breaking and resealing of just one of the strands of DNA.
DNA topoisomerase I
are a family of drugs that block the action of topoisomerases.
Quinolones
kills bacteria by inhibiting DNA gyrase
Nalidixic acid
Inhibitors of eukaryotic topoisomerase II
(etoposide, teniposide) are becoming
useful as anticancer agents.
- Replication is completed when the two replication forks meet each other on the side of the circle opposite the origin.
are repetitive sequences at the ends of linear
DNA molecules in eukaryotic chromosomes
Telomeres
Cancer cells often have relatively high levels of
telomerase, preventing the telomeres from
becoming shortened and contributing to the
immortality of malignant cells.
- autosomal recessive disorder,
characterized by extreme sensitivity
to sunlight, skin freckling and
ulcerations, and skin cancer. - most common deficiency occurs in
the excinuclease enzyme
Xeroderma pigmentosum
- deficiency in the ability to repair
mismatched base pairs in DNA that
are accidentally introduced during
replication
Hereditary nonpolyposis colorectal cancer
- encodes a kinase essential for p53
activity - inactivated in ataxia telangiectasia
- characterized by hypersensitivity to
x-rays and predisposition to
lymphomas
ATM gene
- refers to the length of DNA that is
replicated following one initiation event
Replicon
Three main features of the DNA
synthesis reaction:
- DNA polymerase I catalyzes formation of
phosphodiester bond between 3’-OH of the
deoxyribose (on the last nucleotide) and the 5’-
phosphate of the dNTP. - DNA polymerase “finds” the correct
complementary dNTP at each step in the
lengthening process. - Direction of synthesis is 5’ to 3’
