biochem synthesis of dna
TRANSCRIPT
AP Biology 2007-2008
Synthesis of DNA
June.21.2010
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 aComplementary strand.
AP Biology
Each molecule generated by the replication process contains
one intact parental strand and one newly synthesized strand.
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.
AP Biology
Cultured MDCK cells
Day 1 Day 3
AP Biology
Watson and Crick1953 article in Nature
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
AP Biology
Directionality of DNA You need to
number the carbons! it matters!
OH
CH2
O
4
5
3 2
1
PO4
N base
ribose
nucleotide
This will beIMPORTANT!!
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
PO4
base
CH2
O
base
OPO
C
O–O
CH2
1
2
4
5
1
2
3
3
4
5
5
Sounds trivial, but…this will beIMPORTANT!!
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
AP Biology
Bonding in DNA
….strong or weak bonds?How do the bonds fit the mechanism for copying DNA?
3
5 3
5
covalentphosphodiester
bonds
hydrogenbonds
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
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
AP Biology
DNA Replication Large team of enzymes coordinates replication
Let’s meetthe team…
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
AP Biology
DNAPolymerase III
Replication: 2nd step
But…We’re missing something!
What?
Where’s theENERGY
for the bonding!
Build daughter DNA strand add new
complementary bases DNA polymerase III
AP Biology
energy
ATPGTPTTPCTP
Energy of ReplicationWhere does energy for bonding usually come from?
ADPAMPGMPTMPCMPmodified nucleotide
energy
We comewith our ownenergy!
And weleave behind anucleotide!
YourememberATP!
Are there other ways
to get energyout of it?
Are thereother energynucleotides?You bet!
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
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 53
DNAPolymerase III
DNAPolymerase III
DNAPolymerase III
DNAPolymerase III
energy
energy
energy
Replication energy
3
3
5B.Y.O. ENERGY!The energy rules
the process
5
AP Biology
energy
35
5
5
3
need “primer” bases to add on to
energy
energy
energy
3
no energy to bond
energy
energy
energy
ligase
3 5
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
53
53 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
AP Biology
DNA polymerase III
Replication fork / Replication bubble
5
3 5
3
leading strand
lagging strand
leading strand
lagging strandleading strand
5
3
3
5
5
3
5
3
5
3 5
3
growing replication fork
growing replication fork
5
5
5
5
53
3
5
5lagging strand
5 3
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 53 5 3
growing replication fork
primase
RNA
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
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!
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
TTAAGGGTTAAGGGTTAAGGG
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
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 Kornberg1959
Roger Kornberg2006
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
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
AP Biology
1
2
3
4
What does it really look like?
AP Biology 2007-2008
Any Questions??
AP Biology
energy
ATPGTPTTPATP
Energy of ReplicationWhere does energy for bonding usually come from?
ADPAMPGMPTMPAMPmodified nucleotide
We comewith our ownenergy!
And weleave behind anucleotide!
YourememberATP!
Are there other ways
to get energyout of it?
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 53
DNAPolymerase III
Replication energy
3
3
5B.Y.O. ENERGY!The energy rules
the process
5
AP Biology
5
3
3
5
35
3 5
no energyto bond
AP Biology
energy
5
3
3
5
35
3 5
ligase
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!
AP Biology
Replication fork
3’
5’
3’
5’
5’
3’
3’ 5’
direction of replication
AP Biology
DNA synthesis in prokaryotes
1. Replication is bidirectional.
2. Replication is semi-conservative.
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.
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.
AP Biology
以上です。