DNA/RNA and DNA Replication Flashcards
Francis Crick, a graduate student, and an American postdoctoral fellow James Watson joined forces in late 1951 at the Cavendish Laboratory at the University of Cambridge and began working together on the structure of DNA.
•They used the data from several groups to build their model of DNA: (2)
–Observations of Erwin Chargaff on base composition of DNA
–X-ray diffraction studies of Rosalind Franklin (and Maurice Wilkins)
Watson’s and Crick’s model of DNA was based on X-ray — — of Rosalind Franklin (at King’s College)
diffraction data
Watson’s and Crick’s Model Provides and explanation for Chargaff’s Rule
of A=T and G=C
In Prokaryotic organisms the DNA is organized in a linear or contiguous fashion and transcription of the DNA into RNA results in a RNA copy that is ready for use as a
template for protein synthesis (translation)
In Prokaryotic organisms the RNA transcript can be translated into a protein during the transcription process as there is no
nucleus
In Eukaryotic organisms the DNA is broken up into regions or blocks of sequence that will give rise to the
protein sequence (coding regions or exons)
These exons are separated by (2)
regions that do not code for protein (introns) and regions at the 5’ and 3’ ends that do not encode protein called untranslated regions (UTRs)
In Eukaryotic organisms one strand of the DNA is first copied in a — fashion and then the introns are removed by a process called —
linear
splicing
Subsequent modifications take place that give rise to the mature mRNA, which is transported out of the nucleus for use as the
temple for protein synthesis (translation)
In Eukaryotic organisms primary transcripts are often spliced in multiple combinations of exons known as
alternative splicing
alternative splicing gives rise to a family of possible proteins that can have slightly different (3)
functions, regulation and/or tissue specificity (i.e. different splice variants are found in different tissues)
Linear DNA must be Condensed in order to
fit into a Cell or Nucleus
in prokaryotes, DNA is condensed by a set of — and proteins in
polyamines
back and forth loops
in eukaryotes, DNA is first condensed into
nucleosomes
each nucleosome involves (2)
~200 bp of DNA and a set of core histone proteins
Nucleosomes look like a “beads on a string” and are usually packaged together to give
chromatin fiber structure
Chromatin exists in what two forms?
euchromatin and heterochromatin
Euchromatin (2)
a more relaxed structure and transcriptionally active
Heterochromatin (2)
more highly condensed and generally not transcriptionally active
Chromatin can be further condensed into (3 steps)
solenoids then supersolenoids ultimately into chromosomes by function specific sets of proteins
bacterial vs eukaryotic chromosomes
bacteria have a single major heritable unit or chromosome
DNA in eukaryotic cells is packaged into several chromosomes
Bacteria can also have separate smaller DNA entities called
plasmids
eukaryotes also have DNA genomes in their
mitochondria
plants have a DNA genome in their
chloroplasts
how many protein coding genes?
~20,000-25,000
The average gene is about — bp long, contains about — exons and a coding sequence of about — bp
27,000
9
1340
The full set of proteins or proteome is more complex, it is estimated that the average gene gives (2)
8 isoforms, splice variants, etc.
Human Genome: ~ – billion bp
3.2
Mutation that can cause disease: – bp
1
Differences between siblings: ~ – million bp
1-2
Difference between unrelated humans: ~ – million bp
6
Humans vs. Chimps: ~ – million bp different
50
Humans vs. Mice: ~ – million bp different
100
— orientation of the strands
Antiparallel
Strands are —
complementary
Each strand can act as a — for the synthesis of a new strand
template
Central Dogma of Genetic Information Flow
DNA to RNA to protein
DNA polymerase catalyzes the stepwise addition of a
deoxyribonucleotide-triphosphate to the 3’-OH end of a polynucleotide chain, the primer strand, that is paired to a second template strand
The newly synthesized DNA strand is synthesized in the
5’-to-3’ direction
The reaction is driven by a large, favorable free-energy change, caused by the
release of pyrophosphate and its subsequent hydrolysis to molecules of inorganic phosphate
The shape of a DNA polymerase molecule, as determined by x-ray crystallography, roughly looks like a
right hand in which the palm, fingers, and thumb grasp the DNA and form the active site
DNA polymerase synthesizes the new strand in the —direction
5’ to 3’
DNA polymerase requires a template strand and a strand to build off of with a
free 3’-OH group
DNA polymerase polymerase activity vs exonuclease activity
5’- 3’ polymerase activity
3’- 5’ exonuclease activity
DNA polymerases are a self-correcting enzymes that
remove their own polymerization errors
DNA polymerases have what kind of activity during DNA synthesis
3’-to-5’ exonuclease proofreading
DNA synthesis initiates in AT rich regions known as
origins of replication
In the early 1960s, studies on whole replicating chromosomes revealed a localized region of replication that moves progressively along the
parental DNA double helix
Because of its Y-shaped structure, this active region is called a
replication fork
the replication fork contains a multi-enzyme/protein complex that contains the
DNA polymerase synthesizes the DNA of both new daughter strands
Leading strand
Daughter strand that is synthesized continuously
Lagging strand
Daughter strand that is synthesized discontinuously
Because both daughter DNA strands are synthesized in the 5’-to-3’ direction, the DNA synthesized on the lagging strand must be made initially as a series of shortDNA molecules, called
Okazaki fragments
On the lagging strand, the Okazaki fragments are synthesized sequentially, with those nearest the fork being the
most recently made
DNA synthesis begins following DNA unwinding and RNA primer synthesis at
replication origins
Lagging Strand DNA Synthesis begins with the synthesis of a
short RNA primer by a special nucleotide-polymerizing enzyme
A schematic view of the reaction catalyzed by
DNA primase
DNA primase
the enzyme that synthesizes the short RNA primers made on the lagging strand using DNA as a template
Unlike DNA polymerase , this DNA primase can start a
new polynucleotide chain
Unlike DNA polymerase , this DNA primase can start a new polynucleotide chain by
joining two nucleoside triphosphate together
The primase synthesizes a short polynucleotide in the 5’-to-3’ direction and then stops, making the 3’ end of this primer available for the
DNA polymerase that produce Okazaki fragment
RNA primer length
about 10 nt
A reason why RNA primer is required for DNA synthesis…
Self-correcting polymerase, such as DNA polymerases, cannot start chains
Lagging Strand DNA Synthesis multiple DNA fragments are generated as
DNA synthesis proceeds
Lagging Strand DNA Synthesis: Finally lagging strand fragments are joined to form a
copied double-stranded DNA
The reaction catalyzed by
DNA ligase
DNA ligase seals a broken
phosphodiester bond
DNA ligase uses a molecule of — to activate the 5’ end at the nick before forming the new bond
ATP
In this way, the energetically unfavorable nick-sealing reaction is driven by being coupled to the
energetically favorable process of ATP hydrolysis
DNA replication is very accurate to prevent
errors in DNA from passing on to the next generation
Human genome is approximately
3 x 109 base pairs
Only - nucleotide changes at each time of cell division
3
The number of cells in our human body is
10^13 (10 trillion)
error rate of
1 per 10^9 bases
5’ to 3’ polymerization results in error of
1 in 10^5
3’ to 5’ exonucleolytic proofreading results in error rate of
1 in 10^2
strand directed mismatch repair results in error rate of
1 in 10^2
Compared to an error rate of 1 per 10^9 bases, there is still high chances to undergo
nucleotide changes
DNA helicases
Hydrolyse ATP and change the shape of a protein, move rapidly along a DNA strand; where they encounter a region of double helix, they continue to move along their strand, thereby, prying apart the helix.
Special proteins help open up the DNA double helix in front of
the replication
Single-strand DNA-binding proteins (helix-destabilizing proteins)
Bind to exposed DNA strands
Unable to open a long DNA helix directly, but aid helicases by
stabilizing the unwound helix
Cooperative binding completely coats and straightens out the regions of single-stranded DNA on the lagging strand template, thereby preventing formation of
the short hairpin helix that would otherwise impede synthesis by the DNA polymerase
A — —holds a moving DNA polymerase onto the DNA
sliding ring
The regulated — — that holds DNA polymerase on the DNA
sliding clamp
the clamp loader dissociates into solution once the
clamp has been assembled
At a true replication fork, the clamp loader remains close to the lagging-strand polymerase, ready to
assemble a new clamp at the start of each new Okazaki fragment
Werner syndrome
A premature aging disease
Mutations in RECQL2 gene, which encodes a homolog of E.coli RecQ
DNA helicase
Cells with an altered Werner protein may (2), causing
divide more slowly or stop dividing earlier than normal
growth problem
the altered protein may allow
DNA damage to accumulate
the altered protein may allow DNA damage to accumulate, which could
impair normal cell activities and cause the health problems associated with this condition
