Landmark Experiments in DNA Replication
Requirements of ALL DNA Polymerases
Mechanism of DNA Replication
DNA Polymerase I of E. coli and its activities
I. Why Study DNA Replication?
1) To understand cancer—uncontrolled cell division (DNA replication)
2) To understand aging—cells capable of finite # of doublings
3) To understand diseases related to DNA repair
a) Bloom’s Syndromeb) Xeroderma Pigmentosumc) Werner’s Syndrome
Keith Richards (of the Rolling Stones)
Example of premature agingNOT caused by a hereditary disease
II. Landmarks in the Study of DNA ReplicationA. 1953 Watson and Crick:
B. 1958 Meselson and Stahl
From the structure of DNA they predicted that the DNA strands could act as templates for the synthesis of new strands: base complementarity
Three Potential DNA Replication Models
New DNAOld DNA
The Meselson-Stahl Experiment“The most beautiful experiment in biology”
Conclusion: DNA is Replicated Semiconservatively:1. The parental strands separate during DNA replication.2. Daughter DNA molecules consist of one new and one old (parental) strand.
Parental
III. General Features of DNA Replication
1. require a DNA template and a primer with a 3’ OH end (DNA polymerases can only elongate; no de novo initiation of DNA synthesis)
2. require dNTPs
3. synthesize DNA in a 5’ to 3’ direction.
All DNA Polymerases:
Short RNA primers are needed for initiation in vivo
4. require metal ions (Mg2+ or Zn2+) as cofactors
The Substrates for DNA Replicationdivalent
The Mechanism of DNA Synthesis
1
2
1
DNA Synthesis Is Exergonic
dNTP + (dXMP)n (dXMP)n+1 + P~P G = -3.5 kcal/mole
P~P 2 P G = -7 kcal/mole
Total: dNTP + (dXMP)n (dXMP)n+1 + 2 P G = -10.5 kcal/mole
2
Replication of the E. coli Chromosome is Semidiscontinuous
Replicates continuously
DNA synthesis is going in same direction as replication fork
Because of the anti-parallel structure of the DNA duplex, new DNA must be synthesized in the direction of fork movement in both the 5’ to 3’ and 3’ to 5’ directions overall.
Replicates discontinuously
DNA synthesis is going in opposite direction as replication fork
However all known DNA polymerases synthesize DNA in the 5’ to 3’ direction only.
The solution is semidiscontinuous DNA replication.
Joined by DNA ligase
IV. DNA Polymerase I
The first DNA polymerase was discovered by Arthur Kornberg in 1957 → DNA Polymerase I ofE. coli
A. . E coli DNA Pol I has 3 enzymatic activities:
1) 5’ → 3’ DNA polymerase
2) 3’ → 5’ exoncuclease (For proofrea )ding
3) 5’ → 3’ DNA exonucleas (e For repa :ir toedit outsecti ons of damage dDNA)
1 323
Hans Klenow showed tha t limited proteolysis wit h eithe r subtilisi n o r trypsi n will cle avePo l Iint otwo biologicall y active fragments.
Facts abou t DNA Synthesis Erro r Rate :s—DNA polymera seinserts one incorrec tnucleoti defor eve 10ry 5 nucleotides added.—Proofreading exonucleas es decrease the appearance of an incorrect paired base t o one in every107 nucleotides added.—Actua l error rate observed i n a typical cell is one mistake i n every 1010 nucleotides adde .d—Error rat efor RNA Polymerase is 1/105 nucleotides.
aa 928Klenow Fragment
5’ to 3’ E .xo 5’ to 3’ Pol & 3’ to 5’ ExoN C
Klenow Fragment
DNA Repair(Errors fixed after DNA replication)
No Proofreading
Nick Translation
5’ 3’ 3’ 5’
5’ 3’ 3’ 5’
Newly synthesized. DNA
DNA Polymerase I
+
5’-dNMPs
nick
nick
5’ 3’ exonuclease activity digests DNA5’ 3’ polymerase activity replaces the digested DNA with new DNA
They act together to edit out sections of damaged DNA
The 5’ to 3’ Exonuclease and 5’ to 3 Polymerase of Pol I Result in “NickTranslation”:
5’ 3’ 3’ 5’
I
5’ 3’ 3’ 5’
5’ → 3’ exonuclea se edits damage dDNA
Newly synthesized. DNA
DNA Polymerase I
+
5’ -dNMPs
nick
nick
B. Processivity
DNA Polymerases Can be Processive or Distributive
Processivity is continuous synthesis by polymerase without dissociation from thetemplate.
Processive Polymerization
A DNA polymerase that is Distributive will dissociate from the template after eachnucleotide addition.
Distributive Polymerization
1 nucleotide
Used inDNAReplication
Suitable forDNA Repair
Proc. Dist.
How to Measure Processivity
[32P]-dNTPs
ssDNAtemplate
M13
Mg2+
5 min. @ 37oC
STOP w/ EDTA
DNA Pol
Polyacrylamide Gel
Processivity experimentsrequire a large excess oftemplate to Pol toprevent reassociation tothe same template.
primer
–
+
DNA Pol I
RNA
Okazaki fragment
>10 kb
1 kb
Roles of DNA Pol III and Pol I in E. coli
Pol III—main DNA replication enzyme. It exists as a dimer to coordinate the synthesis of both the leading and lagging strands at the replication fork.
Pol I—repair enzyme to remove RNA primers that initiate DNA synthesis on both strands. It is need predominantly for maturation of Okazaki fragments.
1) Removes RNA primers (5’3’ Exo)2) Replaces the RNA primers with DNA (5’3’ Pol & 3’5’ Exo proofreading)
Q: Why do Okazaki fragments initiate with RNA primers?
A: Because DNA polymerases require a primer but can’t synthesize them de novo
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