What are the properties of prokaryotic DNA?
- 2 complementary, antiparallel strands
- Semiconservative replication
- Helical conformation
- Covalently closed circle
- Supercoiled loops
What topological problems result from circular DNA and supercoiled loops?
- Pulling apart supercoiled DNA strands at the replication fork
- Greater coiling ahead of fork
- Bunched up and prevent unwinding - Replicated strands will be intertwined and must be separated
- Newly replicated DNA must be partitioned into daughter cells
What is the solution to DNA getting bunched up behind the replication fork?
DNA gyrase (topoisomerase II)
- Continuously removes supercoils by breaking and resealing dsDNA
What are the three stages of DNA replication?
- Initiation
- Elongation
- Termination
What are the steps of initiation for DNA replication?
- Formation of open complex
- Formation of pre-priming complex
- Formation of replication fork (replisome)
Explain the formation of the open complex.
- DnaA –> binds oriC
- HU (histone-like protein) activates ATP-dependent reaction that unwinds the A-T-rich region at the 5’ end of the origin sequence
- SSB (single strand binding proteins) coat ssDNA
Explain the creation of the pre-priming complex.
- Opening is unwound by DnaB (helicase)
- Bi-directional –> 2 replication forks form
Where in the cell does the formation of the replication fork take place?
In the cytoplasm in a “replisome”
What is the initiation site?
Origin = oriC locus
What proteins are involved in elongation (DNA replication)?
- DNA polymerase
- DnaG (aka primase)
- Sliding clamps
- Clamp loader
How many DNA polymerases does E. coli have?
5
What is the function of DNA polymerase I?
Attaches Okazaki fragments
What is the function of DNA polymerase II, IV, and V?
Repairs damaged DNA
What is the function of DNA polymerase III?
Replicates DNA at the fork
What is the function of DnaG?
Synthesizes the RNA primer for the Okazaki fragments (perhaps also the RNA primer for the leading strand)
What is the function of sliding clamps?
Tethers DNA pol to the DNA template
What is the function of the clamp loader?
Load sliding clamps on
- Tethered to DNA pol –> interacts with clamps
- Stimulates the release of Okazaki fragments
Where does DNA replication stop?
Stops at region of chromosome opposite oriC = Ter
What makes Ter sites unusual?
- Ter sites allow replication to proceed in only 1 direction
- Several Ter sites in termination zone facing both directions
What is the function of the Tus protein?
Tus protein binds to Ter site and imposes one-way travel by inhibiting DnaB (replicative helicase)
- Binds with sequence specificity –> gives Ter its directionality
What are the two mechanisms of chromosome separation?
- None or equal recombination events occur –> chromosomes are linked non-covalently
- Topoisomerase IV separates sister chromosomes via decatenation - Unequal number of recombination events occur –> covalently linked sister chromosomes
- Must be separated by recombination
- Occurs at dif sites
Explain chromosome partitioning.
- Prokaryotes don’t have a spindle apparatus
- Proteins compact the chromosomes and pull/guide origins to the poles
When does septum formation begin?
Begins soon after completion of DNA replication
What is the first morphological sign of cell division?
Septum formation
