Prokaryotic DNA Replication Nov. 12, 2004 MBB 407/511 Lecture 19: Prokaryotic DNA Replication (Part I)

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 Example of premature aging NOT caused by a hereditary disease a) Bloom’s Syndrome b) Xeroderma Pigmentosum c) Werner’s Syndrome Keith Richards (of the Rolling Stones)

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.” All hybrids 1/2 old: 1/2 new 1/2 hybids: 1/2 new 1/4 old: 3/4 new All hybrids Three potential DNA replication models and their predicted outcomes The actual data!

III. General Features of DNA Replication DNA Synthesis: 1. requires a DNA template and a primer with a 3’ OH end. (DNA synthesis cannot initiate de novo) Short RNA molecules act as primersin vivo 2. requires dNTPs. 3. occurs in a 5’ to 3’ direction.

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 Replicates discontinuously DNA synthesis is going in opposite direction as replication fork Joined by DNA ligase 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. However all known DNA polymerases synthesize DNA in the 5’ to 3’ direction only. The solution is semidiscontinuous DNA replication.

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

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

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) >10 kb DNA Pol I 1 kb RNA Okazaki fragment RNA primer replaced with DNA by Pol I’s nick translatiton activity Okazaki fragment