CELL AND MOLECULAR BIOLOGY
BIOL30095
Prepared by: Coleen Gail C. Pareja BSBIO 3-4MB
CENTRAL DOGMA OF BIOLOGY
TOPIC OUTLINE
• DNA and RNA Structure and Function
• DNA Replication
• DNA to RNA to Protein
Genetic Material
- Double-stranded DNA is a right-handed helix of
paired, complementary, antiparallel strands, each
composed of an ordered string of nucleotides bearing
A (adenine), T (thymine), G (guanine), or C (cytosine).
Characteristics of Genetic Material
- Replication– the genetic material of cells replicates
and is doubled in amount
- Storage of information– molecule/s acts a reposito-
ry of genetic information that may or may not be ex-
pressed.
- Expression of information- central dogma of molec-
ular genetics: “DNA makes RNA, which makes pro-
teins”
- Variation by mutation– genetic material is also the
source of variability among organisms
DNA Replication
• Conservative replication produces one helix
containing entirely old DNA and other helix
containing entirely new DNA.
• Semiconservative model produces two heli-
ces, and each has one strand of old DNA and
one strand of new DNA.
• Dispersive model produces DNA helices in
which each strand has alternating segments of
new and old DNA.
Chemical Nature of Genetic Material
• Gregor Mendel first postulated the existence of
genes
• Johann Friedrich Miescher reported the dis-
covery of the substance now known as
“nuclein” (DNA) in 1869,
• Walther Flemming first observed chromo-
somes as he studied dividing cells under the
microscope.
• Frederick Griffith: Bacterial transformation
- Frederick Griffith conducted a series of experiments
using Streptococcus pneumoniae bacteria and mice.
- R strain. When grown in a petri dish, the R bacteria
formed colonies, or clumps of related bacteria, that
had well-defined edges and a rough appearance
(hence the abbreviation "R"). The R bacteria were
nonvirulent, meaning that they did not cause sickness
when injected into a mouse.
- S strain. S bacteria formed colonies that were
rounded and smooth (hence the abbreviation "S").
The smooth appearance was due to a polysaccharide,
or sugar-based, coat produced by the bacteria. This
coat protected the S bacteria from the mouse immune
CENTRAL DOGMA OF BIOLOGY
system, making them virulent (capable of causing dis-
ease). Mice injected with live S bacteria developed
pneumonia and died.
- Griffith tried injecting mice with heat-killed S bacte-
ria (that is, S bacteria that had been heated to high
temperatures, causing the cells to die). Unsurprisingly,
the heat-killed S bacteria did not cause disease in
mice.
- The experiments took an unexpected turn, however,
when harmless R bacteria were combined with harm-
less heat-killed S bacteria and injected into a mouse.
Not only did the mouse develop pneumonia and die,
but when Griffith took a blood sample from the dead
mouse, he found that it contained living S bacteria.
- Conclusion: R-strain bacteria must have taken up
what he called a "transforming principle" from the heat
-killed S bacteria, which allowed them to "transform"
into smooth-coated bacteria and become virulent.
• Avery, MacLeod, and McCarty Experiment
- Several lines of evidence suggested to Avery and his
colleagues that the transforming principle might be
DNA:
A. The purified substance gave a negative result in
chemical tests known to detect proteins, but a strongly
positive result in a chemical test known to detect DNA.
B. The elemental composition of the purified trans-
forming principle closely resembled DNA in its ratio of
nitrogen and phosphorous.
C. Protein- and RNA-degrading enzymes had little ef-
fect on the transforming principle, but enzymes able to
degrade DNA eliminated the transforming activity.
- Conclusions: These results all pointed to DNA as the
likely transforming principle.
• The Hershey-Chase experiments
- Hershey and Chase studied bacteriophage, or virus-
es that attack bacteria. The phages they used were
simple particles composed of protein and DNA, with
the outer structures made of protein and the inner core
consisting of DNA.
- They found that when phages infect a host bacterium,
the phages first attach themselves to the outside of the
bacterium. Then, a piece of the phage enters the bac-
terium and subsequently replicates itself inside the
cell.
- Conclusion: The main criteria that was done for the
experiment was that both DNA and protein contains
phosphorus. Explanation: this experiment there was
evidence that the DNA was responsible for the genetic
characteristics of an individual and not the protein.
DNA: Structure and Function
• Deoxyribonucleic acid (DNA) molecule consists
of two long polynucleotide chains composed of
four types of nucleotide subunits (Chains = DNA
Chain or DNA strand)
- Base Composition: DNA is a nucleotide consisting of
the deoxyribose to which one phosphate is esterified
at the 5 position of the sugar ring and one nitrogenous
base is attached at the 1 site.
- Nitrogenous bases are present in a nucleic acid:
A. Pyrimidines- contain a single ring contains two
different pyrimidines, thymine (T) and cytosine
(C) [PyCuT]
B. Purines- contain two rings DNA two different
purines, guanine (G) and adenine (A). [PurGA]
- Nucleotide has a polarized structure: one end, where
the phosphate is located, is called the 5’ end, while
the other end is the 3’ end.
• Chargaff’s Rules
Chargaff discovered the following rules of DNA base
composition:
Chargaff's rules state that (1) DNA from any species of
any organism should have a 1:1 stoichiometric ratio of
purine and pyrimidine bases (i.e., A+G=T+C ) and,
(2) that the amount of guanine should be equal to cyto-
sine and the amount of adenine should be equal to
thymine.
• Franklin’s X-Ray Diffraction Data
- X-ray diffraction analysis indicated that the distance
between the nucleotides of the stack was 3.4 Å (0.34
nm) and suggested the presence of a large structural
repeat every 3.4 nm.
CENTRAL DOGMA OF BIOLOGY
• Watson and Crick Model - DNA and histones are organized into repeating sub-
- Deoxyribonucleic Acid (DNA) is a double-stranded, units called nucleosomes.
helical molecule. It consists of two sugar-phosphate - A nucleus 10 mm in diameter can pack 200,000 times
backbones on the outside, held together by hydrogen this length of DNA within its boundaries.
bonds between pairs of nitrogenous bases on the in- - Packing ratio of the DNA in nucleosomes is approxi-
side. The bases are of four types (A, C, G, & T): pairing mately 7:1.
always occurs between A & T, and C & G. - Assembly of the 30-nm fiber increases the DNA-
- realized that these pairing rules meant that either packing ratio to 40:1.
strand contained all the information necessary to make - Mitotic chromosomes represent the ultimate in chro-
a new copy of the entire molecule, and that matin compactness with a ratio of 10,000:1.
the aperiodic order of bases might provide a "genetic
• Heterochromatin and Euchromatin
code".
- Euchromatin returns to a dispersed state after mito-
- Watson and Crick shared the Nobel Prize in 1962 for
sis.
their discovery, along with Maurice Wilkins (1916 -
2004), who had continued research to provide a large - Heterochromatin is condensed during interphase.
body of crystallographic data supporting the model. - Constitutive heterochromatin remains condensed
- Working in the same lab, Rosalind Franklin (1920 - all the time. Found mostly around centromeres and
1958) had earlier produced the first clear crystallo- telomeres. Consists of highly repeated sequences and
graphic evidence for a helical structure. few genes.
- The Importance of the Watson-Crick Proposal - Facultative heterochromatin is inactivated during
certain phases of the organism’s life. Is found in one of
1. Storage of genetic information
the X chromosomes as a Barr body through X inactiva-
2. Replication and inheritance tion. X inactivation is a random process, making adult
3. Expression of the genetic message females genetic mosaics.
• DNA Supercoiling - Facultative heterochromatin is inactivated during
- When the DNA helix has the normal number of base certain phases of the organism’s life. Is found in one of
pairs per helical turn it is in the relaxed state. the X chromosomes as a Barr body through X inactiva-
- If DNA is in the form of a circular molecule, or if the tion. X inactivation is a random process, making adult
ends are rigidly held so that it forms a loop, then females genetic mosaics.
overtwisting or undertwisting leads to the supercoiled
state.
- Supercoiling occurs when the molecule relieves the
helical stress by twisting around itself.
- Overtwisting leads to positive supercoiling.
- Undertwisting leads to negative supercoiling.
- Twist can be altered in a circular model by breaking
the circle, over or undertwisting and then reconnect-
ing the ends.
• Chromosomes and Chromatin
- Chromosomes consist of chromatin fibers, com-
posed of DNA and associated proteins.
- Each chromosome contains a single, continuous DNA
molecule.
- Nucleosomes– basic unit of chromatin structure
This “beads-on-a-string” appearance led to the suggestion that the beads
consist of protein (presumably histones) and the thin filaments connecting the
beads correspond to DNA. We now refer to each bead, along with its associat-
ed short stretch of DNA, as a nucleosome.
CENTRAL DOGMA OF BIOLOGY– PRACTICE QUESTIONS
1. In the late 19th and early 20th century, most scien-
tists believed that was the genetic material because it
offers more potential complexity.
PROTEIN Scientists wondered which component of a
chromosome carried hereditary information: protein or
DNA. There are 20 different amino acids for building a
protein polymer, while DNA polymers are made of only
four nucleotide bases. Because proteins appeared for
more complex, most scientists of the day believed that
protein carried genetic information within the chromo-
some.
2. Frederick Griffith made his important observations
about "transformation" while attempting to develop a
vaccine against which pathogenic organism?
S. PNEUMONIAE During the First World War, hun-
dreds of thousands of servicemen died from pneumonia,
caused by the bacterium Streptococcus pneumonia (or
S. pneumonia). In the early 1920s, Frederick Griffith be-
gan studying the bacteria in hopes of developing a vac-
cine against it. Although he was unable to develop a vac-
cine for pneumonia, he made one of the most important
discoveries in the field of biology: "transformation.
3. When Frederick Griffith coined the term
"transformation," this was in reference to the genetic
re- programming of
STRAINS OF BACTERIA Dr. Griffith mixed living R
bacteria (which are not pathogenic) with heat-killed S
bacteria (that causes pneumonia) and injected the mix
into mice, who developed pneumonia. The harmless live
R strain was somehow transformed into the pathogenic S
strain. This discovery showed that organisms can be ge-
netically "re-programmed" into a slightly different ver-
sion of themselves.
4. In the famous Avery, MacLeod, and McCarty experi-
ment, the tube that was treated with resulted in the de-
struction of the transforming material
DNase The scientists added different enzymes to test
tubes to see which could prevent R strain bacteria from
transforming into the S strain. Each enzyme would de-
stroy one possible transforming agent: RNA protein,
DNA, lipids, and carbohydrates. The tube that was treat-
ed with the enzyme to break down DNA lost the ability to
transform the R Strain into the S strain, while the others
were unaffected.
5. The Hershey and Chase experiment served as pow-
erful independent confirmation that DNA was indeed
the genetic material. They made their discovery using
which type of organism?
BACTERIOPHAGE For their experiment, Hershey and
Chase used on extremely small virus called a bacterio-
phage (or just phage), which only infects bacterial cells.
When these phage inject a bacterial cell, they somehow
“reprogram" the bacterium produce more phage.
6. As determined by Frederick Griffith's experiment,
when DNA is "transformed"
It produces a slightly different organism. Dr. Grif-
fith's experiment showed that organisms can be genet-
ically “reprogrammed” into a slightly different version
of themselves. In Griffith's experiment, the harmless R
strain of S pneumoniae bacteria was transformed into
the pathogenic S strain of S. pneumoniae bacteria, pre-
sumably because of the transfer of genetic material
from a donor (the S strain).
7. After Griffith's results were made known, scientists
all over the world began to repeat his experiment in
slightly different ways. Which statement BEST ex-
plains why that happened?
Scientists wanted to build on and extend his re-
search. Although it was clear that a genetic transfor-
mation occurred, the question remained: Which mole-
cule is the transforming agent? Protein, RNA, DNA, li-
pids, and carbohydrates were all possibilities, and sci-
entists needed to experiment further to find out exactly
what was happening during genetic transformation.
CENTRAL DOGMA OF BIOLOGY
• RNA: Structure and Function
- Ribonucleic acid (RNA) is made up of nucleotides
containing the sugar ribose.
- Bases: adenine (A), *uracil (U), cytosine (C), and gua-
nine (G)
- RNA is a helper to DNA, allowing protein synthesis to
occur according to the genetic information that DNA
provides
- Types of RNA
1. mRNA- Nucleus, migrates to ribosomes to cyto-
plasm; carries DNA sequence information to ri-
bosome
2. tRNA- Cytoplasm; provides linkage between
mRNA and amino acids; transfers amino acids to
ribosomes
3. rRNA- Cytoplasm; structural component of ribo-
somes

• DNA Replication and Repair

CENTRAL DOGMA OF BIOLOGY
• DNA Replication: semiconservative Replica-
tion
- DNA replication takes place by separation of the
strands of the double helix, and synthesis of two
daughter strands complementary to the two parental
templates
- Watson and Crick envisioned that replication oc-
curred by gradual separation of the strands of the dou-
ble helix (zipper).
- Semiconservative because each daughter duplex con-
tains one strand from the parent structure.
- DNA replication is called semiconservative because
half of the parent structure is retained in each of the
daughter duplexes.
- Two Other Types of Replication (in the absence of in-
formation on the mechanism responsible for replica-
tion)
1. Conservative Replication- one of the daughter
duplexes would contain only parental DNA,
while the other daughter duplex would contain
only newly synthesized DNA.
2. Dispersive Replication- the daughter duplexes
would contain strands that were composites of
old and new DNA.
• Replication of Circular DNA
- Replication begins at a specific site in the DNA
called the origin of replication (oriC)
- DNA replication is bidirectional from the origin of
replication.
- The process of DNA replication in prokaryotes can
be summarized as follows:
1. DNA unwinds at the origin of replication.
2. Helicase opens up the DNA-forming replication
forks; these are extended in both directions.
3. Single-strand binding proteins coat the DNA
around the replication fork to prevent rewind-
ing of the DNA.
4. Topoisomerase binds at the region ahead of the
replication fork to prevent supercoiling (over-
winding).
5. Primase synthesizes RNA primers complemen-
tary to the DNA strand.
6. DNA polymerase III starts adding nucleotides to
the 3′-OH (sugar) end of the primer.
7. Elongation of both the lagging and the leading
strand continues.
8. RNA primers are removed and gaps are filled
with DNA by DNA pol I.
9. The gaps between the DNA fragments are
sealed by DNA ligase.
• Resolving DNA Replication Issues
- Helicase, SSBs, supercoiling, gyrase, and topoiso-
merase– for unwinding the DNA Helix
- Primase– (RNA primer) for initiation of DNA synthe-
sis
• Continuous and Discontinuous DNA Synthe-
sis
- Since the two strands of double-helical DNA run in
opposite (antiparallel) directions, continuous synthe-
sis of two new strands at the replication fork would
require that one strand be synthesized in the 5′ to 3′
direction while the other is synthesized in the oppo-
site (3′ to 5′) direction.
- Continuous DNA synthesis occurs in the 3' 5' direction
on the parent strand. This is often referred to as the
leading strand with new nucleotides being added to
the 3' end.
CENTRAL DOGMA OF BIOLOGY
- Okazaki fragments are short sequences of DNA nu-
cleotides (approximately 150 to 200 base pairs long in
eukaryotes) which are synthesized discontinuous-
ly and later linked together by the enzyme DNA ligase
to create the lagging strand during DNA replication.
- Discontinuous DNA synthesis occurs in the 5' 3' direc-
tion on the parent strand. This strand is often referred
to as the lagging strand.
• DNA Polymerases
- The polymerase molecules responsible for construc-
tion of the two new strands of DNA both move in a 3’-to
-5’ direction along the template, and both construct a
chain that grows from its 5’-P terminus.
- See Table 17.1
- DNA polymerase III- enzyme that synthesizes DNA
strands during replication in E. coli, is part of a large
“replication machine” called the DNA polymerase III
holoenzyme
- βclamp– one of the noncatalytic components of the
holoenzyme that keeps the polymerase associated
with the DNA template.
- Contrasting properties of DNA polymerases:
(1) they must remain associated with the template
over long stretches if they are to synthesize a
continuous complementary strand
(2) they must be attached loosely enough to the
template to move from one nucleotide to the
next.
* These contrasting properties are provided by the
doughnut-shaped beta clamp that encircles the DNA
and slides along it. As long as it is attached to a β
“sliding clamp,” a DNA polymerase can move proces-
sively from one nucleotide to the next without diffusing
away from the template
- The polymerase on the leading-strand template re-
mains tethered to a single βclamp during replication.
- When the polymerase on the lagging-strand template
completes the synthesis of an Okazaki fragment, it dis-
engages from the β clamp and is cycled to a new β
clamp that has been assembled at an RNA primer–DNA
template junction located closer to the replication fork.
• DNA Replication: DNA polymerase as an ex-
onuclease
Exonuclease activities of DNA polymerase I: 5’ → 3’ and
3’ → 5’ functions
- DNA polymerase I is involved in DNA repair and also
removes RNA primers and replaces them with DNA.
- Exonucleases degrade nucleic acids by removing 5’
or 3’ terminal nucleotides.
- 5’-3’ exonuclease function removes approximately
10 nucleotides from the 5’ end of a single strand nick.
This activity plays a key role in removing the RNA pri-
mer.
- 3’-5’ exonuclease function removes mispaired nucle-
otides from the 3’ end of the growing DNA. This func-
tion is key in maintaining the accuracy of DNA synthe-
sis.
• Ensuring High Fidelity during DNA Replica-
tion
- During proofreading, mismatched bases are ex-
cised.
- The 3 → 5 exonuclease activity removes approxi-
mately 99 out of every 100 mismatched bases, raising
the fidelity to about 10-7 –10-8 .
- Bacteria possess a mechanism called mismatch re-
pair that operates after replication and corrects nearly
all of the mismatches that escape the proofreading
step. Together these processes reduce the overall
observed error rate to about 10-9.
- The fidelity of DNA replication can be traced to three
distinct activities:
(1) accurate selection of nucleotides
(2) immediate proofreading, and
(3) Post-replicative mismatch repair. Replication is
rapid (~103 nucleotides/sec).
• Replication in Eukaryotes
1. Helicases unwind the parental double helix.
2. Single-strand binding proteins stabilize the un-
wound parental DNA.
3. The leading strand is synthesized continuously
in the 5’ -> 3’ direction by DNA polymerase III.
4. The lagging strand is synthesized discontinu-
ously. Primase synthesizes a short RNA primer,
which is extended by DNA polymerase to form
an Okazaki fragment.
CENTRAL DOGMA OF BIOLOGY
5. After the RNA primer is replaced by DNA (by anoth- 2. Base excision repair removes a variety of altered
er DNA polymerase), DNA ligase joins the Okazaki nucleotides that produce minor distortions in the DNA
fragment to the growing strand. helix.
Cells possess a variety of glycosylases that recognize
• DNA Replication: Chromatin structure and remove various types of altered bases. Once the
base is removed, the remaining portion of the nucleo-
tide is removed by an endonuclease, the gap is en-
larged by a phosphodiesterase activity, and the gap is
filled and sealed by a polymerase and ligase.
3. Mismatch repair is responsible for removing in-
correct nucleotides incorporated during replication
that escape the proofreading activity of the polymer-
ase. In bacteria, the newly synthesized strand is se-
lected for repair by virtue of its lack of methyl groups
compared to the parental strand.
(Histones that were present in parental nucleosomes
prior to replication are indicated in blue; newly synthe- 4. Double-strand breaks are repaired as proteins bind
sized histones are indicated in red.) to the broken strands and join the ends together. An
- The parental (H3H4)2 tetramers remain intact and are alternate pathway of DSB repair, called homologous
recombination, is not discussed.
distributed randomly to both daughter duplexes.
- In contrast, the pairs of H2A/H2B dimers present in
parental nucleosomes separate and recombine ran- • In addition to the classic DNA polymerases
domly with the (H3H4)2 tetramers on the daughter du- involved in DNA replication and repair, cells
plexes. also possess an array of DNA polymerases
- The assembly of DNA into nucleosomes is a rapid that facilitate replication at sites of DNA le-
event. sions or misalignments. These polymerases,
which engage in translesion synthesis, lack pro-
- Histones remain intact during replication and old and
cessivity and proofreading capability and are
new histones are distributed randomly between the
more error-prone than classic polymerases.
two daughter duplexes.
- The assembly of nucleosomes is facilitated by a net-
work of accessory proteins.
- CAF-1 protein is a histone chaperone that interacts
with PCNA
• DNA Repair
- DNA is subject to damage by many environmental
influences, including ionizing radiation, common
chemicals, and ultraviolet radiation.
- Four major types of DNA repair systems:
1. Nucleotide excision repair (NER) systems operate
by removing a small section of a DNA strand contain-
ing a bulky lesion, such as a pyrimidine dimer.
During NER, the strands of DNA containing the lesion are
separated by a helicase; paired incisions are made by
endonucleases; the gap is filled by a DNA polymerase;
and the strand is sealed by a DNA ligase. The template
strands of genes that are actively transcribed are prefer-
entially repaired by NER.
CENTRAL DOGMA OF BIOLOGY– PRACTICE QUESTIONS
1.What must happen first in order for DNA replication 16. During replication, what enzyme adds complimen-
to occur? tary bases?
- DNA is unwound. - polymerase
2. Okazaki fragments form on the: 17. The sugar in RNA is ____, the sugar in DNA is ____.
- lagging strand - ribose, deoxyribose
3. What is required for DNA replication to occur? 18. Amino acids are joined together into a protein
- DNA helicase, DNA ligase, DNA polymerase chain by _____.
4. A nucleotide consists of: - transfer RNA
- a nitrogen base, sugar, and phosphate 19. Proteins contain ______ different amino acids,
5. The element that transformed the bacteria in Grif- whereas DNA and RNA are composed of ___ different
fith’s experiments was nucleotides.
- DNA - 20, 4
6. The Hershey-Chase research showed that: 20. Okazaki fragments occur on the ___ and are bond-
ed together by _____.
- DNA was the molecule of heredity
- lagging strand, ligase
7. The “rungs” of the DNA ladder are made of:
- Bases
8. Before the Hershey-Chase experiment, many scien-
tists believed that ___ carried the hereditary info
- proteins
9. In order to transform a virulent form of bacteria, non
-encapsulated bacteria must:
- be exposed to killed capsulated bacteria
10. A DNA strand has the following bases: A A G C C
A. What are the bases on its complementary strand?
-TTCGGT
11. In the Hershey Chase Experiment, DNA was la-
beled with ______, and bacteriophage protein was la-
beled with ______.
- radioactive phosphorus, radioactive sulfur
12. What is the best description for the arrangement of
the sides of the DNA molecule?
- antiparallel
13. If a DNA molecule is found to be composed of 40%
thymine, what percentage of guanine would be ex-
pected?
- 10%
14. The enzymes that break hydrogen bonds and un-
wind DNA are:
- helicases
15. DNA replication results in:
- 2 DNA molecules that each contain a strand of the
original.