MBB 407/511

Lecture 19:Prokaryotic DNA Replication

(Part I)

Nov. 12, 2004

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

I. Why Study DNA Replication?

1) To understand cancer

2) To understand aging

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. Historical Background

A. 1953 Watson and Crick: DNA Structure Predicts a Mechanism of Replication“It has no escaped our notice that the specific pair we have postulated immediately suggests a possible copying mechanism for the genetic material.”

B. 1958 Meselson and Stahl: DNA Replication is Conservative

The Meselson-Stahl Experiment“the most beautiful experiment in biology.”

Three potential DNA replication models and their predicted outcomes The actual data!

1/4 old:3/4 new

1/2 hybids:1/2 new

Allhybrids

1/2 old:1/2 new

Allhybrids

III. General Features of DNA Replication

1. requires a DNA template and a primer with a 3’ OH end. (DNA synthesis cannot initiate de novo)

2. requires dNTPs.

3. occurs in a 5’ to 3’ direction.

DNA Synthesis:

Short RNA moleculesact as primersin vivo

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 Polymerases of E. coli

The first DNA polymerase was discovered by Arthur Kornberg in 1957 → DNAPolymera se I

A. . E coli DNA Pol I has 3 enzymatic activities:

1) 5’ → 3’ DNA polymeraseKlenow Fragment

2) 3’ → 5’ exoncuclease (For proofrea )ding

3) 5’ → 3’ DNA exonucleas (e To edit out sections of damaged DN )A

1 323

Hans Klenow showed tha t limited proteolysis wit h eithe r subtilisi n o r trypsi n will cleavePol I into t wo biologicall yacti vefragments.

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 onei ne 10very 7 nucleotides added.—Actua l error rate observed i n a typical cell is one mistake i n every 1010 nucleotidesadded.—Error rat efor RNA Polymerase is 1/105 nucleotides.

aa 928Klenow Fragment

5’ to 3’ E .xo5’ to 3’ Pol & 3’ to 5’ Exo

Model for the Interaction of Klenow Fragment with DNA

How the Proofreading Activity of Klenow Fragment Works

DNA Polymerase I can Perform “Nick Translation”

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 dissociationfrom the template.

A DNA polymerase that is Distributive will dissociate from the templateafter each nucleotide addition.

Processive Polymerization

Distributive Polymerization

1 nucleotide

Used inDNAReplication

Suitable forDNA Repair

Proc. Dist.

How to Measure Processivity

dATPdCTP

dGTP[32P]-dTTP

ssDNAtemplate

M13Mg2+

5 min. @ 37oC

STOP w/ EDTA

DNA Pol

Polyacrylamide Gel

Processivity experimentsrequire a large excess oftemplate to Pol to preventreassociation to the sametemplate.

primer

DNA Pol III is highly “processive” DNA Pol I is” distributive”

Pol I & II – main DNA repair enzymePol III – main DNA replication 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)

RNA primer replaced withDNA by Pol I’s nick translatiton activity

Okazaki fragment