3. Recall…
•DNA and RNA are nucleic
acids
•An important macromolecule in
organisms that stores and carries
genetic information
4. What is the Double Helix?
•Shape of DNA
•Looks like a twisted
ladder
•2 coils are twisted
around each other
•Double means 2
•Helix means coil
5. The Structure of DNA
• Made out of nucleotides
•Includes a phosphate group,
nitrogenous base and 5-carbon
pentose sugar
Nucleotide
Structure
1
“link
” in a
DNA
chai
n
7. The Structure of DNA
Backbone = alternating P’s and sugar
•Held together by COVALENT bonds
(strong)
•Inside of DNA molecule = nitrogen
base pairs
•Held together by HYDROGEN
bonds (weaker)
Backbon
e
8. Phosphodieste
r Bond
The covalent
that holds
together the
backbone
Found between
P &
deoxyribose
sugar
10. DNA is antiparallel
Antiparallel means that
the 1st
strand runs in
a 5’ 3’ direction and
the 2nd
3’ 5’
direction
THEY RUN IN
OPPOSITE or
ANTIPARALLEL
DIRECTIONS
P end is 5’ end (think: “fa”
sound)
-OH on deoxyribose
sugar is 3’ end
5’ and 3’ refers to the carbon #
on the pentose sugar that P or
11. DNA in Cells
2 broad categories of cells
1. Eukaryotic cells: have
nucleus with DNA
DNA is contained in
structure called a
chromosome
Chromosomes are a
LINEAR (line) shape with
ENDS called telomeres
(protective “caps”)
2. Prokaryotic cells: no
nucleus (nucleoid region
instead) which contains
DNA
DNA is a CIRCULAR
12. DNA Bonding
Purines (small word, big base)
Adenine
Guanine
Pyrimidines
(big word, small base)
Cytosine
Thymine
Chargaff’s rules
A=T, C=G
Hydrogen BondsHydrogen Bonds attractions between
the stacked pairs; WEAK bonds
13. Why Does a Purine Always
Bind with A Pyrimidine?
14. DNA Double Helix
http://www.sumanasinc.com/webc
ontent/animations/content/DNA_st
ructure.html
Watson & Crick said that…
strands are complementary; nucleotides line up
on template according to base pair rules
(Chargaff’s rules)
A to T and C to G
LET’S PRACTICE…
Template: 5’AATCGCTATAC3’
Complementary strand: 3’ TTAGCGATATG5’
16. DNA Replication
DNA Replication =
DNA DNA
Parent DNA makes
2 exact copies of
DNA
Why??
Occurs in Cell
Cycle before
MITOSIS so
each new cell
can have its
own FULL copy
of DNA
17. 17
Replication FactsReplication Facts
DNA has to be copiedDNA has to be copied
before a cell dividesbefore a cell divides
DNA is copied during theDNA is copied during the SS
or synthesis phase ofor synthesis phase of
interphaseinterphase
New cells will needNew cells will need identicalidentical
DNA strandsDNA strands
copyright cmassengale
18. 18
Synthesis Phase (S phase)Synthesis Phase (S phase)
S phase during interphase of the
cell cycle
Nucleus of eukaryotes
Mitosis
-prophase
-metaphase
-anaphase
-telophase
G1 G2
S
phase
interphase
DNA replication takesDNA replication takes
place in the S phase.place in the S phase.
copyright cmassengale
19. Four requirements for DNA to
be genetic material
Must carry information
Cracking the genetic code
Must replicate
DNA replication
Must allow for information to change
Mutation
Must govern the expression of the
phenotype
Gene function
20. Much of DNA’s sequence-specific information is
accessible only when the double helix is unwound
Proteins read the DNA sequence of nucleotides as the
DNA helix unwinds.
Proteins can either bind to a DNA sequence, or initiate
the copying of it.
• Some genetic information is accessible even in intact,
double-stranded DNA molecules
• Some proteins recognize the base sequence of DNA
without unwinding it (One example is a restriction enzyme).
DNA stores information in the
sequence of its bases
21. DNA replication occurs with greatDNA replication occurs with great
fidelityfidelity
Somatic cell DNA stability and reproductive-cellSomatic cell DNA stability and reproductive-cell
DNA stability are essential. Why?DNA stability are essential. Why?
Pan troglodytes
99% sequence identity
Identity
Genetic diseases
Homo sapiens sapiens
99.9% sequence identity
22. DNA Replication
Process of duplication of the entire genome
prior to cell division
Biological significance
extreme accuracy of DNA replication is
necessary in order to preserve the integrity of
the genome in successive generations
In eukaryotes , replication only occurs during
the S phase of the cell cycle.
Replication rate in eukaryotes is slower
resulting in a higher fidelity/accuracy of
replication in eukaryotes
23. Basic rules of
replication
A. Semi-conservative
B. Starts at the ‘origin’
C. Synthesis always in the 5-3’ direction
D. Can be uni or bidirectional
E. Semi-discontinuous
F. RNA primers required
26. DNA Replication: a closer
look
http://henge.bio.miami.edu/m
allery/movies/replication.mov
27. DNA Replication
Steps:
Initiation
involves assembly of replication
fork (bubble) at origin of
replication
sequence of DNA found at a
specific site
Elongation
Parental strands unwind and
daughter strands are synthesized.
the addition of bases by proteins
Termination:
the duplicated chromosomes
separate from each other. Now,
there are 2 IDENTICAL copies of
28. Segments of single-stranded DNA are called
template strands.
Copied strand is called the complement
strand (think “c” for copy)
BEGINNING OF DNA REPLICATION
(INITIATION)
Gyrase (type of topoisomerase)
relaxes the supercoiled DNA.
DNA helicase (think “helix”)
binds to the DNA at the replication fork
untwist (“unzips”) DNA using energy from ATP
Breaks hydrogen bonds between base pairs
Single-stranded DNA-binding
proteins (SSBP)
stabilize the single-stranded template DNA during
the process so they don’t bond back together.
29. base pairs
5’
5’
3’
3’
percoiled DNA relaxed by gyrase & unwound by
helicase
Helicase
ATP
SSB Proteins
http://media.pearsoncmg.com/bc/
bc_campbell_biology_7/media/inte
SSB Proteins
Gyrase
30. (Elongation)
After SSBP’s bind to each template…
RNA Primase binds to helicase
primase is required for DNA synthesis
Like a “key” for a car ignition
makes a short RNA primers
Short pieces of RNA needed for DNA
synthesis
DNA polymerase
adds nucleotides to RNA primer makes
POLYNUCLEOTIDES (1st
function)
After all nucleotides are added to compliment
strand…
RNA primer is removed and replaced
with DNA by DNA polymerase (2nd
function)
DNA ligase
“seals” the gaps in DNA
Connects DNA pieces by making phosphodiester bonds
32. DNA Polymerase
Leading strand
base pairs
5’
5’
3’
3’
percoiled DNA relaxed by gyrase & unwound by
helicase + proteins:
Helicase
ATP
SSB Proteins
RNA Primer
primase
2DNA Polymerase
1
RNA primer
replaced by DNA
Polymerase & gap
is sealed by
ligase
Gyrase
33. Starts at origin
Initiator proteins identify specific base
sequences on DNA called sites of origin
Prokaryotes – single origin site E.g E.coli - oriC
Eukaryotes – multiple sites of origin (replicator)
E.g. yeast - ARS (autonomously replicating
sequences)
Prokaryotes Eukaryotes
34. Replication Origin
Site where replication begins
1 in E. coli
1,000s in human
Strands are separated to allow
replication machinery contact with
the DNA
Many A-T base pairs because easier toMany A-T base pairs because easier to
break 2 H-bonds that 3 H-bondsbreak 2 H-bonds that 3 H-bonds
Note anti-parallel chains
dna A (20-50 monomers) binds to the
origin of replication and is also called as origin-
binding protein.
This requires ATP and results in separation
(melting) of two strands of DNA.
The two complementary strands of DNA separate
at the site of replication to form a bubble.
36. Elongation
Antiparallel nature:
Sugar
(3’end)/phosphate (5’
end) backbone runs
in opposite directions
one strand runs 5’
3’,
other runs 3’ 5’
DNA polymerase only
adds nucleotides at
the free 3’ end of
NEW STRAND
forming new DNA
strands in the
5’ 3’ direction
38. does DNA replication only occur in the 5’ to 3’ directdoes DNA replication only occur in the 5’ to 3’ direct
Should be PPP here
39.
40. Elongation (con’t)
Leading (daughter) strand
NEW strand made toward the
replication fork (only in 5’ 3’
direction from the 3’ 5’
master strand
Needs ONE (1) RNA
primer (about 5-50
nucleotides, variable with species)
made by Primase
This new leading strand is
made CONTINOUSLY
41. Elongation (con’t)
Lagging (daughter) strand
NEW strand synthesis away from
replication fork
Replicate DISCONTINUOUSLY
Creates Okazaki fragments
Short pieces of DNA
Okazaki fragments joined by DNA
ligase
“Stitches” fragments together
Needs MANY RNA primer
made by Primase
42. 3
DNA Polymerase
5’ →
3’
Leading strand
base pairs
5’
5’
3’
3’
percoiled DNA relaxed by gyrase & unwound by
helicase + proteins:
Helicase
ATP
SSB Proteins
RNA Primer
primase
2
DNA Polymerase
Lagging strand
Okazaki
Fragments
1
RNA primer replaced by
DNA Polymerase & gap is
sealed by DNA ligase
Gyrase
43.
44. Termination
(Telomeres) Telomeres
Short repeats of “G” base found at END
of LINEAR chromosomes in
eukaryotes
protect ends of linear chromosomes
The repeated sequence of GGGTTA
make up the human telomeres.
Telomerase is the enzyme that
makes telomeres.
45. Telomeres, Aging &
Cancer
Telomeres get shorter as cell
divides leads to aging???
Most cancers come from body cells.
Cancers cell have ability to divide
indefinitely.
Normal cells limited to ~50-75
divisions stop making
telomerase.
85–90% cancer cells continue to
make high levels of telomerase &
are able to prevent further
shortening of their telomeres.
46. TOPOISOMERASES
There are several types of topoisomerases:
1. Type I Topoisomerases:
Reversibly cuts a single-strand of the double helix and subsequently reseals the
same.
In prokaryotes, catalyze relaxation of the negative supercoils where as in eukaryotes relax
both the negative as well as the positive supercoils.
2. Type II Topoisomerases:
Multimeric enzymes, i.e. they cleave both the strands and reseal them.
Require ATP.
Isolated from bacteria are called gyrases.
Both prokaryotic and eukaryotic type II topoisomerases relax negative as well as positive
supercoils.
3. Type III Topoisomerases:
Bacterial enzymes with type I properties, i.e. they relax supercoils without ATP.
Remove circular DNA products called catenates which are generated just prior to the
completion of DNA replication.
REVERSE GYRASES:
Unusual type of topoisomerases that have been isolated from various species of
archaebacteria.
Introduce positive supercoils into DNA and protect it from the denaturating
conditions such as high temperature and acidity.
47. INHIBITORS OF TOPOISOMERASES:
i) Antibiotics of the quinolone category:
E.g. norfloxacin, ciprofloxacin, ofloxacin, nalidixic acid etc. and
some anticancer agents such as doxorubicin, adriamycin and
etoposide, block topoisomerases II activity. Thus arrest DNA replication as
well as RNA transcription.
ii) Nucleotide analogs:
e.g. 6-mercaptopurine, 5-flurouracil.
iii) Others:
E.g. camptothecin, anthracycline, etc. interfere only with the enzyme
catalyzed resealing of the DNA strands. They do not affect the overall
activity of the enzyme but convert these topoisomerases into the DNA-
breaking agents.
Since DNA degradation leads to cell death, these drugs are used in the
treatment of certain haematological neoplasms, e.g. leukemias and
lymphomas.
48. DNA POLYMERASES
Three distinct forms of the enzyme are found in
prokaryotes.
1. DNA Polymerase I (Pol I):
Structurally, a single polypeptide.
Important role in DNA replication as well as its repair
in E.coli.
Has three distinct activities, i.e.
5’→ 3’ polymerase (synthetic) activity, and 3’→ 5’ as well as
5’→3’ exonuclease (hydrolytic) activities.
Has proof reading activity.
49. 2. DNA Polymerase II (Pol II):
Has 5’ 3’ polymerase as well as 3’ 5’ exonuclease
activities but lacks 5’ 3’ exonuclease activity.
Mainly participates in DNA repair.
3. DNA Polymerase III (Pol III):
Can polymerize a DNA strand as well as edit its
mistakes but lacks nick-translation.
50. REPLICATION IN EUKARYOTES
The replication on the leading strand of DNA is rather simple, involving DNA
polymerase δ and a sliding clamp called proliferating cell nuclear
antigen (PCNA).
PCNA forms a ring around DNA to which DNA polymerase δ binds.
Formation of this ring also requires another factor namely replication
factor C (RFC).
The parental strands of DNA are separated by the enzyme helicase.
A single-stranded DNA binding protein called replication protein A (RPA)
binds to the exposed single-stranded template.
The enzyme primase forms a complex with DNA polymerase α which
initiates the synthesis of Okazaki fragments.
The primase activity of pol α-primase complex is capable of producing 10-bp
RNA primer.
The enzyme activity is then switched from primase to DNA polymerase α
which elongates the primer by the addition of 20-30
deoxyribonucleotides.
Thus, by the action of pol α –primase complex, a short stretch of DNA
attached to RNA is formed.
And now the complex dissociates from the DNA.
51. The next step is the binding of replication factor C (RFC) to the
elongated primer (short RNA-DNA).
RFC serves as a clamp loader, and catalyses the assembly of
proliferating cell nuclear antigen (PCNA) molecules.
The DNA polymerase δ binds to the sliding clamp and elongates the
Okazaki fragment to a final length of about 150-200 bp.
By this elongation, the replication complex approaches the RNA primer of
the previous Okazaki fragment.
The RNA primer removal is carried out by a pair of enzymes namely
Rnase H and flap endonuclease I (FENI).
This gap created by RNA removal is filled by continued elongation of the
new Okazaki fragment (carried out by polymerase δ).
The small nick that remains is finally sealed by DNA ligase.
Eukaryotic DNA is tightly bound to histones (basic proteins) to form
nucleosomes which, in turn, organize into chromosomes.
The DNA strands separate for replication, and the parental histones
associate with one of the parental strands.
As the synthesis of new DNA strand proceeds, histones are also produced
simultaneously, on the parent strand.
At the end of replication, of the two daughter chromosomal DNAs formed,
one contains the parental histones while the other has the newly
synthesized histones.
Notes de l'éditeur
06_27_humans_whales.jpg
Figure 6.16
06_15_proofreading.jpg
Right handed supercoiling = negative supercoiling (underwinding)
Left handed supercoiling = positive supercoiling