Biochem synthesis of dna

1. AP Biology 2007-2008
Synthesis of DNA
June.21.2010

2. AP Biology
DNA synthesis occurs by the process of replication.
During replication, each of the two parental strands of
DNA serves as a template for the synthesis of a
Complementary strand.

3. AP Biology
Each molecule generated by the
replication process contains
one intact parental strand and one newly
synthesized strand.

4. AP Biology
In eukaryotes, DNA replication occurs during the
S phase of the cell cycle
The cell divides during the next phase (M), and
each daughter cell receives an exact copy of the DNA of the parent cells.

5. AP Biology
Cultured MDCK cells
Day 1 Day 3

6. AP Biology
Watson and Crick
1953 article in Nature

7. AP Biology
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

8. AP Biology
Directionality of DNA
You need to
number the
carbons!
it matters!
OH
CH
2
O
4¢
5¢
3¢
2¢
1¢
PO
4
N base
ribose
nucleotide
This will be
IMPORTANT!!

9. AP Biology
The DNA backbone
Putting the DNA
backbone together
refer to the 3¢ and 5¢
ends of the DNA
the last trailing carbon
OH
O
3¢
PO
4
base
CH
2
O
base
O
P
O
C
O
–
O
CH
2
1¢
2¢
4¢
5¢
1¢
2¢
3¢
3¢
4¢
5¢
5¢
Sounds trivial, but…
this will be
IMPORTANT!!

10. AP Biology
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¢

11. AP Biology
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

12. AP Biology
Base pairing in DNA
Purines
adenine (A)
guanine (G)
Pyrimidines
thymine (T)
cytosine (C)
Pairing
A : T
2 bonds
C : G
3 bonds

13. AP Biology
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

14. AP Biology
DNA Replication
Large team of enzymes coordinates replication
Let’s meet
the team…

15. AP Biology
Replication: 1st step
Unwind DNA
helicase enzyme
unwinds part of DNA helix
stabilized by single-stranded binding proteins
single-stranded binding proteins
replication fork
helicase

16. AP Biology
DNA
Polymerase III
Replication: 2nd step
But…
We’re missing
something!
What?
Where’s the
ENERGY
for the bonding!
Build daughter DNA
strand
add new
complementary bases
DNA polymerase III

17. AP Biology
energy
ATPGTPTTPCTP
Energy of Replication
Where does energy for bonding usually come from?
ADPAMPGMPTMPCMP
modified nucleotide
energy
We come
with our own
energy!
And we
leave behind a
nucleotide!
You
remember
ATP!
Are there
other ways
to get energy
out of it?
Are there
other energy
nucleotides?
You bet!

18. AP Biology
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

19. AP Biology
Adding bases
can only add
nucleotides to
3¢ end 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
Replication energy
3¢
3¢
5¢
B.Y.O. ENERGY!
The energy rules
the process
5¢

20. AP Biology
energy
3¢5¢
5¢
5¢
3¢
need “primer” bases to add on to
energy
energy
energy
3¢
no energy
to bond
energy
energy
energy
ligase
3¢ 5¢


21. AP Biology
Limits of DNA polymerase III
can only build onto 3¢ end of
an existing DNA strand
Leading & Lagging strands
5¢
5¢
5¢
5¢
3¢
3¢
3¢
5¢
3¢
5¢
3¢
3¢
Leading strand
Lagging strand
Okazaki fragments
ligase
Okazaki
Leading strand
continuous synthesis
Lagging strand
Okazaki fragments
joined by ligase
“spot welder” enzyme
DNA polymerase III


3¢
5¢
growing
replication fork

22. AP Biology
DNA polymerase III
Replication fork / Replication bubble
5¢
3¢
5¢
3¢
leading strand
lagging strand
leading strand
lagging strand
leading strand
5¢
3¢
3¢
5¢
5¢
3¢
5¢
3¢
5¢
3¢
5¢
3¢
growing
replication fork
growing
replication fork
5¢
5¢
5¢
5¢
5¢
3¢
3¢
5¢
5¢
lagging strand
5¢
3¢

23. AP Biology
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¢
5¢
3¢
3¢
3¢
5¢
3¢
5¢
3¢5¢
3¢
growing
replication fork
primase
RNA

24. AP Biology
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¢
5¢
3¢
3¢
3¢
3¢
growing
replication fork
DNA polymerase I
RNA
ligase

25. AP Biology
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¢
5¢
3¢
3¢
3¢
3¢
growing
replication fork
DNA polymerase I
RNA
Houston, we
have a problem!

26. AP Biology
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
TTAAGGG

27. AP Biology
Replication fork
3’
5’
3’
5’
5’
3’
3’ 5’
helicase
direction of replication
SSB = single-stranded binding proteins
primase
DNA
polymerase III
DNA
polymerase III
DNA
polymerase I
ligase
Okazaki
fragments
leading strand
lagging strand
SSB

28. AP Biology
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

29. AP Biology
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

30. AP Biology
Fast & accurate!
It takes E. coli <1 hour to copy
5 million base pairs in its single
chromosome
divide to form 2 identical daughter cells
Human cell copies its 6 billion bases &
divide into daughter cells in only few hours
remarkably accurate
only ~1 error per 100 million bases
~30 errors per cell cycle

31. AP Biology
1
2
3
4
What does it really look like?

32. AP Biology 2007-2008
Any Questions??

33. AP Biology
energy
ATPGTPTTPATP
Energy of Replication
Where does energy for bonding usually come from?
ADPAMPGMPTMPAMP
modified nucleotide
We come
with our own
energy!
And we
leave behind a
nucleotide!
You
remember
ATP!
Are there
other ways
to get energy
out of it?

34. AP Biology
Adding bases
can only add
nucleotides to
3¢ end of the
growing DNA strand
need a primer
nucleotide to
bond to
strand grows 5¢®3¢
DNA
Polymerase III
Replication
energy
3¢
3¢
5¢
B.Y.O. ENERGY!
The energy rules
the process
5¢

35. AP Biology
5¢
3¢
3¢
5¢
3¢5¢
3¢ 5¢
no energy
to bond


36. AP Biology
energy
5¢
3¢
3¢
5¢
3¢5¢
3¢ 5¢
ligase

37. AP Biology
Loss of bases at 5¢ ends
in every replication
chromosomes get shorter with each replication
limit to number of cell divisions?
DNA polymerase III
DNA polymerases can
only add to 3¢ end of
an existing DNA
strand
Chromosome erosion
5¢
5¢
5¢
5¢
3¢
3¢
3¢
3¢
growing
replication fork
DNA polymerase I
Houston, we
have a problem!

38. AP Biology
Replication fork
3’
5’
3’
5’
5’
3’
3’ 5’
direction of replication

39. AP Biology
DNA synthesis in prokaryotes
1.Replication is bidirectional.
3.Replication is semi-conservative.

40. AP Biology
Bidirectional replication of a circular chromosome
Replication begins at the point of origin (oriC) and
proceeds in both directions at the same time.

41. AP Biology
Unwinding of Parental Strands
Topoisomerases: can break phosphodiester bonds and rejoin them
relieve the supercoiling of the parental duplex caused by unwinding.
DNA gyrase is a major topoisomerase in bacterial cells.

42. AP Biology
以上です。