C.   Replication- General Features

1. Bidirectional

a.  Origin(s) of Replication- proceeds in both directions

b.     Replication fork- where DNA helix splits into 2 ssDNA

2. Unwinding DNA

a. DNA Gyrase- (Type II Topoisomerase)- introduces negatives coils with ATP

-type I cuts only ssDNA, type II cuts dsDNA

b. Helicase- Unwinds the DNA by breaking the hydrogen bonds

-disrupts H-bonds instead of breaking phosphodiester bonds (gyrase)

-requires ssDNa for binding (see replication process)

c.     SSB (single stranded DNA-binding protein) bonds with single stranded to prevent the double helix from recombining.

d.     Primase- binds to open DNA and synthesizes an RNA primer

- Once for leading strand, many times on lagging strand

e.      DNA polymerase- builds the ìdaughter strandî from the parent strand template

-builds only in the 5í to 3í direction

-needs to have the 3í OH exposed to add new nucleotide bases

1.  Leading strand- parent strand that is 3í ý 5í direction, copied continuously

2.  Lagging strand- copied in a discontinuous mode, short segments

-Okazaki fragments- ~1000 to 2000 nucleotides in length

f.       DNA Ligase

-responsible for joining Okazaki fragments together on the lagging strand

D.   DNA Polymerase

1. Properties

a.      selects the appropriate nucleotide base that complements template bases

b.     builds in the 5íý3í direction and is antiparallel to the template

c.     cannot initiate DNA synthesis- requires an oligonucleotide prime with free 3íOH

2. Types & Function

a. DNA Polymerase I- (from E.coli)

1. Function as polymerase

- able to catalyze DNA synthesis- in vitro- if supplied with deoxynucleotide-5í-triphosphates (dATP, dTTP, dGTP, dCTP), a template DNA strand and a primer

- pyrophosphate is released when a new nucleotide base is added to a pre-existing nucleotide

- falls off about every 20 bases ñ poor processivity

2. Function as exonuclease

-serves as a proof-reader and editor of 3í and 5í ends

 b. DNA Polymerase III

-functions as part of a larger complex- DNA Polymerase III Holoenzyme

-contains 10 subunits that function to increase the processivity- ~4.6 Mbase

-g subunit- clamp loader

-b subunit- sliding clamp

III. Replication- Procedure (E.coli model)

Shockwave video of Replication

A. Initiation

1. Replication begins when DnaA protein attaches to oriC (origin site)

-wraps up the origin and opens the DNA

-the opening of the 45 bp DNA is ATP-dependent

2. DnaB protein binds to the replication fork ý contains helicase and DNA gyrase activity

3. ssBP binds to ssDNA

4. Primase synthesizes primers that are complement to DNA nucleotides

5. DNA polymerase binds to both templates to begin DNA replication

B.    Elongation

1. DnaB advances the replication fork along in 2 directions

2. DNA Polymerase synthesizes new DNA strands along the leading strand in the 5íý3í direction.

-lagging strand has to loop to maintain DNA Polymerase III Holoenzyme complex

3. Primase has to continually produce primers on the lagging strand for each Okazaki fragment

4. DNA Polyermase I excises RNA primers, replaces with DNA nucleotides and edits

5. DNA Ligase seals the nicks between Okazaki fragments

C.   Termination

1. Ter Locus (termination base sequence)

-also requires a Tus protein (replication termination protein)- inhibits the ATP-dependent DnaB helicase activity

2. Topoisomerase II (DNA gyrase) catalyzes the excision of the new doubles helices which splits them for each daughter cells produced during fission

DNA Replication:  Learning Objectives and Guiding Questions