1. DNA Replication Robert F. Waters, Ph.D.

2. Goals:  What is semi-conservative DNA replication?  What carries out this process and how?  How are errors corrected?  What is recombination and how does it occur?

3.  Duplication of DNA for transfer from parental to daughter cells -  DNA replication occurs in a semi- conservative manner –Each strand of DNA acts as a template for synthesis of a new strand –Daughter DNA contains one parental and one newly synthesized strand

4.  Some points to remember about DNA replication: –Three distinct stages: initiation, elongation and termination. –Process carried out by a large complex containing multiple proteins –Process must be rapid (1000 bp/s in E. coli) and accurate - mistakes can be lethal! Repair,  Much of what we know has been determined in E. coli - overall strategy seems to be conserved among organisms

5.  E. coli chromosome is circular, double-stranded DNA (4.6x103 kilobase pairs)  Replication begins at a unique site (origin)  Proceeds bidirectionally until the two replication complexes meet termination site) – then replication stops

6.  Replisome – protein machinery for replication (one replisome at each of 2 replication forks) –New strands of DNA are synthesized at the two replication forks where replisomes are located

7.  Eukaryotic replication –Eukaryotic chromosomes are large linear, double-stranded DNA molecules –Fruit fly large chromosomes ~5.0x104 kb (~10x larger than E. coli) –Replication is still bidirectional –Multiple sites of initiation of DNA synthesis (versus one site in E. coli) –Rate of replication slower than in E. coli – but multiple origins enables rapid replication of genome

8.  Replicating DNA in the fruit fly –Large number of replication forks at opposite ends of “bubbles” of duplicated DNA –evidence for multiple origins of replication

9.  What are the components of replisomes? –The enzyme that adds successive nucleotides to the end of the growing DNA chain is called a DNA polymerase. –E. coli contains three DNA polymerases  DNA polymerase I - repairs DNA and participates in DNA synthesis of one strand  DNA polymerase II - role in DNA repair  DNA polymerase III - the major DNA replication enzyme, responsible for chain elongation

10.  DNA polymerase III –largest of the three polymerases.  Ten different polypeptide subunits. Holoenzyme complex is a dimer –two copies of each subunit.

11.  Chain Elongation Is a Nucleotidyl-Group- Transfer –Base pair between incoming deoxynucleotide 5’ triphosphate (blue) and a residue of the parental strand –Terminal 3’ OH attacks á- phosphorous of incoming nucleotide to form new phosphodiester linkage –Nucleotides added one at a time to the 3’ end –Chain grown is always 5’ to 3’

12.  Some Important points about DNA Polymerase III –Adds one nucleotide at a time –Cannot synthesize DNA is the absence of a template or it the absence of the 3’ end of an existing chain (primer) –DNA polymerase III is a processive enzyme (remains bound to the replication fork until replication is complete) –  -Subunits form a sliding clamp which surrounds the DNA molecule

13.  Sliding clamp of  - subunits of DNA POL III –Two  -subunits associate to form a head-to-tail dimer in the shape of a ring that completely surrounds the DNA –Remaining subunits of DNA pol III are bound to this structure

14.  Proofreading Corrects Polymerization Errors –DNA polymerase III holoenzyme also possesses 3’ 5’ exonuclease activity –Pol III can catalyze both chain elongation and degradation –Recognizes distortion in the DNA caused by incorrectly paired bases –Exonuclease activity removes mispaired nucleotide before polymerization continues –Most uncorrected replication errors are subsequently corrected by the DNA repair enzymes

15. DNA Polymerase Synthesizes Two Strands Simultaneously –DNA pol III catalyzes chain elongation only in the 5’ 3’ direction –Leading strand - synthesized by polymerization in the same direction as fork movement –Lagging strand - synthesized by polymerization in the opposite direction of fork movement –Two core complexes of DNA pol III, one for leading, one for lagging strand - both part of same pol III holoenzyme dimer

16.  Leading strand is synthesized as one continuous polynucleotide –Lagging strand is synthesized discontinuously in short pieces (Okazaki fragments) –Pieces of the lagging strand are then joined by a separate reaction

17.  RNA Primer Begins Each Okazaki Fragment –Primosome is a complex containing primase enzyme which synthesizes short pieces of RNA at the replication fork (complementary to the lagging-strand template) –DNA pol III uses the RNA primer to start the laggingstrand DNA synthesis –Replisome – includes primosome, DNA pol III

18.  Okazaki Fragments Are Joined by Action of DNA Polymerase I and DNA Ligase –Okazaki fragments are joined to produce a continuous strand of DNA in 3 steps:  (1) Removal of the RNA primer - The 5’ 3’ exonuclease activity of DNA pol I  (2) Synthesis of replacement DNA – nick translation: polymerase activity of pol I synthesizes DNA in place of RNA  (3) Sealing of adjacent DNA fragments - DNA  Ligase (Next Process)

19.  Joining of Okazaki fragments by DNA pol I and DNA ligase  Problem: after Okazaki fragment synthesis is complete, have gap between end of Okasaki fragment and RNA primer

20.  DNA pol I extends the Okasaki fragment while its 5 3 exonuclease activity removes the RNA primer.  This is called nick translation.

21.  DNA pol I then dissociates, DNA ligase binds to the nick.

22.  DNA ligase then catalyzes the formation of a phosphodiester linkage between 3’-hydroxyl and 5’- phosphate of adjacent Okazaki fragments – enzyme then dissociates from DNA –Eukaryotic enzymes require ATP cosubstrate –E. coli DNA ligase uses NAD+ as a cosubstrate

23.  The Replisome –Replisome contains: a primosome, DNA polymerase III holoenzyme, additional proteins - assembly into one machine allows coordinated synthesis of leading and lagging strand –DnaB helicase is part of the primosome and facilitates unwinding of the DNA helix –Topoisomerases relieve supercoiling ahead of the replicating fork (not part of the replisome) –Single-stranded binding proteins (SSBs) stabilize single-stranded DNA

24.  Important points about model of replisome action: –1. Lagging strand template loops back through replisome - this way both strands are replicating as replisome moves in one direction. –2. SSB binds the single stranded DNA - replication does not result in large patches of open ssDNA.

25.  As replication proceeds: –helicase unwinds DNA template –replication continues on leading strand –primase makes a new primer on the lagging strand –lagging strand core polymerase finishes an Okasaki fragment

26. –Lagging strand polymerase encounters preceding Okasaki fragment - releases the lagging strand. Nick between Okasaki fragments will be sealed by Pol I and DNA ligase –Polymerase core on leading strand never releases – keeps replication processive

27.  Lagging strand polymerase then binds newly made primer – starts next Okasaki fragment.

28.  Initiation of DNA Replication –Replisome assembles at origin site (oriC in E. coli) –Initial assembly depends on unwinding of the DNA caused by binding certain proteins - often referred to as origin binding proteins –DnaA - origin binding protein in E. coil - helps control replication by controlling initiation frequency

29. –Termination of replication in E. coli occurs at the ter site. –Terminator utilization substance (Tus) binds to the ter site Tus inhibits helicase activity and thus prevents replication forks continuing through this region –Ter site also has sequences that function in separation of daughter chromosomes

30. Proteasome

31. Dynein

32. Metaphase