V. cDNA Libraries (converting mRNA into “complementary DNA”

Removes the RNA part of

RNA:DNA hybrids

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Lecture 19:Prokaryotic DNA Replication

(Part I)

Nov. 11, 2005

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

The Role of Metal Ions In DNA Synthesis

Steric Constraints Prevent Catalysis of rNTPs

Replication of the E. coli Chromosome is Bidirectional

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

“Now this end is called the thagomizer,after the late Thag Simmons.”

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

–

+

Main replicative enzyme Repair enzyme

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