3. Double helix structure of DNA
“It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic
material.” Watson & Crick
4. DNA synthesis
Occur in S phase of the cell cycle
Semiconservative manner
Requires
• DNA template
• dNTPs (dATP, dGTP, dCTP, dTTP)
• DNA Polymerase
• RNA Primer
• ATP
5. Directionality of DNA
You need to
number the
carbons!
it matters!
OH
4
5
CH2
O
3
2
1
PO4
N base
ribose
nucleotide
6. The DNA backbone
Putting the DNA
backbone together
refer to the 3
and 5
ends of the DNA
the last trailing carbon
O
3
PO4
base
2
O
base
–O
1
2
4
1
2
3
OH
C
3
O
P O
O
5
CH2
4
5
CH
5
7. Anti-parallel strands
Nucleotides in DNA
backbone are bonded from
phosphate to sugar
between 3
& 5
carbons
DNA molecule has
“direction”
complementary strand runs
in opposite direction
3
5
5
3
8. Bonding in DNA
….strong or weak bonds?
How do the bonds fit the mechanism for copying DNA?
3
5
3
5
covalent
phosphodiester
bonds
hydrogen
bonds
9. Base pairing in DNA
Purines
adenine (A)
guanine (G)
Pyrimidines
thymine (T)
cytosine (C)
Pairing
A : T
2 bonds
C : G
3 bonds
10. Copying DNA
Replication of DNA
base pairing allows
each strand to serve
as a template for a
new strand
new strand is 1/2
parent template &
1/2 new DNA
17. Energy of Replication
The nucleotides arrive as nucleosides
DNA bases with P–P–P
P-P-P = energy for bonding
DNA bases arrive with their own energy source
for bonding
bonded by enzyme: DNA polymerase III
ATP GTP TTP CTP
18. Replication
Adding bases
can only add
nucleotides to
3end of a growing
DNA strand
need a “starter”
nucleotide to
bond to
strand only grows
5
3
DNA
Polymerase III
DNA
Polymerase III
DNA
Polymerase III
DNA
Polymerase III
energy
energy
energy
3
3
5
5
energy
20.
Strand Separation:
4th step Topoisomerase: enzyme which
relieves stress on the DNA molecule by
free rotation around a single
allowing
strand.
Enzyme
DNA
Enzyme
24. DNA polymerase III
RNA primer
built by primase
serves as starter sequence
for DNA polymerase III
Limits of DNA polymerase III
can only build onto 3
end of
an existing DNA strand
Starting DNA synthesis: RNA primers
5
5
3
3
3
5
3
5
3
5
3
growing
replication fork
primase
RNA 5
25. DNA polymerase I
removes sections of RNA
primer and replaces with
DNA nucleotides
But DNA polymerase I still
can only build onto 3
end of
an existing DNA strand
Replacing RNA primers with DNA
5
5
5
3
3
3
growing
replication fork
DNA polymerase I
RNA 5
3
ligase
26. Loss of bases at 5
ends
in every replication
chromosomes get shorter with each replication
limit to number of cell divisions?
DNA polymerase III
All DNA polymerases can
only add to 3
end of an
existing DNA strand
Chromosome erosion
5
5
5
3
3
3
3
growing
replication fork
DNA polymerase I
RNA 5
27. Repeating, non-coding sequences at the end
of chromosomes = protective cap
limit to ~50 cell divisions
Telomerase
enzyme extends telomeres
can add DNA bases at 5
end
different level of activity in different cells
high in stem cells & cancers -- Why?
telomerase
Telomeres
5
5
5
5
3
3
3
3
growing
replication fork
TTAAGGGTTAAGGG
30. Prokaryotes have circular DNA – no problem at ends
(there aren’t ANY!
Eukaryotes – have special terminal
sequences of 6 nucleotides that repeat from
100-1000 times with no genes included
Telomers
Protect more internal gene materials from
being eroded
Germ cells / sex cells have a special enzyme
(telomerase) that actually restore shortened
telomers
Somatic cells – telomer continues to shorten
and may play a role in aged cell death
Cancer cells
A telomerase prevents very short lengths
34. DNA polymerases
DNA polymerase III
1000 bases/second!
main DNA builder
DNA polymerase I
20 bases/second
editing, repair & primer removal
DNA polymerase III
enzyme
Arthur Kornberg
1959
Roger Kornberg
2006
35.
36.
37.
38. Editing & proofreading DNA
1000 bases/second =
lots of typos!
DNA polymerase I
proofreads & corrects
typos
repairs mismatched bases
removes abnormal bases
repairs damage
throughout life
reduces error rate from
1 in 10,000 to
1 in 100 million bases
39. Structures of DNA polymerase during polymerizing and editing
E: exonucleolytic; P: polymerization
41. Site-directed mismatch repair in eukaryotes
In prokaryotes, old DNAs are usually methylated on A while newly
synthesized ones are not. So Cells can distinguish old and newly
synthesized DNAs and mutate mismatches on new ones.
47. DNA Repair
Spontaneous DNA damage
Pathways to remove DNA damage
Damage detection
The repair of Double-strand break
DNA repair enzymes
55. DNA Repair I
• AP endonuclease
• Deoxyribose phosphate
(DRP) lyase
• POLB (Gap filling)
• Ligase (Nick filling)
56. Base excision repair (BER)
Major pathway for repair of modified
bases, uracil misincorporation, oxidative
damage
Various DNA glycosylases recognize
lesion and remove base at glycosidic
bond, thereby producing an “abasic” or
AP (apurinic/ apyrimidinic) site by base
“flipping out”
One of several AP endonucleases incises
phosphodiesterase backbone adjacent to
AP site
AP nucleotide removed by exonuclease/
dRPase and patch refilled by DNA
synthesis and ligation
61. Types of lesions repaired by BER
Oxidative lesions; 8-oxo-G, highly
mutagenic, mispairs with A, producing GC
--> TA transversions example MutY,
MutM=Fpg from E. coli
Deoxyuracil: from misincorporation of dU
or deamination of dC-->dU, example Ung,
uracil N-glycosylase
Various alkylation products e. g. 3-meA
These lesions are not distorting and do
not block DNA polymerases
Spontaneous depurination (esp. G) yield
abasic sites that are repaired by second
half of BER pathway
62. DNA Repair II
• Incision on either side
• Require helicase to remove
mutated strand
• POLD for leading (gap filling)
POLE for lagging (gap filling)
• Ligase (nick filling)
63. Nucleotide excision repair (NER)
Recognizes bulky lesions that block DNA
replication (i. e. lesions produced by
carcinogens)--example, UV pyrimidine
photodimers
Common distortion in helix
Incision on both sides of lesion
Short patch of DNA excised, repaired by
repolymerization and ligation
In E. coli, mediated by UvrABCD
Many more proteins involved in eukaryotes
Can be coupled to transcription (TCR,
“transcription coupled repair”)
Defects in NER underlie Xeroderma
pigmentosum
64. Xeroderma pigmentosum
• Autosomal recessive mutations in several
complementation groups
•Extreme sensitivity to sunlight
•Predisposition to skin cancer (mean age of skin
cancer = 8 yrs vs. 60 for normal population)
65. Recognition and binding
UvrA acts as classical Nicks delivered
“molecular matchmaker” 3
’
and 5
’to
lesion by UvrBC
Incision Excision and repair
Short fragment
released by
helicase action
66.
67. Site-directed mismatch repair in eukaryotes
In prokaryotes, old DNAs are usually methylated on A while newly
synthesized ones are not. So Cells can distinguish old and newly
synthesized DNAs and mutate mismatches on new ones.
69. Double strand repair 1/2
Nonhomologous end-
joining (NHEJ)
Major pathway for DSB
Require POLM and POLL
Occur in G1 of the cell cycle
Hypersensitive for ionising
radiation in POLM knockout
mice
ligase joins the strands
together
70. Double strand repair 2/2
Homologous end-
joining
damaged site is copied
from the other
chromosome by special
recombination proteins
71. The procedure of general
recombination
DNA synapsis: base pairs form
between complementary strands
from the two DNA molecules
72. Lesion bypass polymerization
Replication-blocking lesions such as UV
photodimers can be repaired by NER but
pose a serious problem if they are in
ssDNA
As a last resort, cells employ “bypass”
polymerases with loosened specificity
In E. coli: DinB (PolIV) and UmuD’C (Pol
V); homologs in eukaryotes; mutated in
XPV
These polymerases are “error-prone” and
are responsible for UV-induced mutation
Expression and function highly regulated:
dependent on DNA damage
73. Characteristics of lesion bypass
polymerases
Error rate 100-10,000 x higher on
undamaged templates
Lack 3’ to 5’ proofreading exonuclease
activity
Exhibit distributive rather than
processive polymerization (nt.
incorporated per binding event)
Support translesion DNA synthesis in
vitro
