DNA Replication ll Flashcards
What 7 proteins and their functions are required for replication in E. coli?
Helicase - unwinds DNA
DNA gyrase - aids in unwinding
Single Strand Binding proteins - ensure DNA doesn’t form secondary structure while unwinding
Primase - adds RNA primer
DNA pol lll - elongation of DNA strand by DNA synthesis
DNA pol l - remove of RNA primer and filling gap with DNA
DNA ligase - links fragments in new DNA strand
What sequence properties are present at the origins of DNA replication? What proteins assists with this?
AT-rich. There are 3 OH bonds (compared to 2 in other pairs). Protien DnaA
What does helicase do? What is its structure? What is another name for it?What is its process driven by?
- DnaB
- Hexameric complex
- Further separates the DNA strands, breaks H-bonds
- Driven bt the free energy of ATP hydrolysis
What protein further facilitates DNA unwinding? What does it prevent?
- DNA gyrase
- A type ll topoisomerase
- prevents DNA supercoiling ahead of the replication fork
What are type l and type ll topoisomerase?
Both work to uncoil DNA as it unwinds. Topoisomerase l cuts a strand uncoils and sticks together. Topoisomerase can cut both strands.
What is a nuclease?
An enzyme that cleave phosphodiester bonds between nucleotides of nucleic acids
When does a Single-stranded binding protein need to stripped away?
Before the ssDNA can be replicated by. DNA polymerase
What does an SSB do?
Single-stranded binding protein has a high affinity to ssDNA and binds to it preventing it from forming a secondary structure, protecting from nucleases
Draw the initiation of replication in E. coli. include; SSB, ATP, Helicase, Gyrase, DNA pol lll, primase
What is a primasome? What are 2 examples?
proteins in E.coli that mediate primer synthesis
- kinds
- Helicase (DnaB)
- Primase (DnaG)
What does primase catalyse? How long is it?
- Polymerisation of RNA segment
- approx 5 - 15 nucleotides in length
- provides the free 3’ OH group
How does the primosome work in the lagging strand?
The primasome is propelled toward the opening of the replication fork (5’ - 3’). It reverses in direction momentarily to synthesise complementary RNA primers for each Okazaki fragment in the lagging strand.
What is the replisome?
- The replisome is a large protein complex that carries out DNA replication, starting at the replication origin.
Why does the replisome have a special “looping arrangement?
allows the replisome to move as a single unit in the direction of the advancing replication fork
Draw the process of the replisome.
What is a holoenzyme?
Enzyme with a whole.
What are the 3 components of DNA pol lll?
- Core enzyme (Pol lll)
- Clamp loader
- B Sliding Clamp
What does the DNA pol lll sliding clamp do? What is its processivity? Draw a diagram of how it works.
- Pol lll not functional until combined with the other components.
- processivity of > 5000
- B subunit forms a ring around the DNA
- sliding clamp can move along it
- keeps pol lll holoenzyme from dissociating
- without would dissociate after 12 residues
What enzymatic activities does DNA Pol l have?
- three different enzymatic activities of DNA pol l
- polymerase domain
- 3-5’ exonuclease
- 5-3’ exonuclease
What does DNA pol l have that DNA pol lll does not?
5’ - 3’ exonuclease domain
What is “Nick Translation”? How are RNA primers removed?
- Single nucleotide removal
- removed and replaced with DNA through the DNA pol l “nick translation” process
- nicks are sealed together by DNA ligase
How does DNA replication stop? What are the 10 Ter terminator sites and how do they act? What protein arrests the replication fork?
- Stop when the terminator sequence reached.
- Requires binding of Tus protein.
Sites start with Ter. A-J
Compare bacterial and eukaryotic DNA replication
- Eukaryotic is similar but much more complex.
- more protein required
- several different modes of replication occur
- have several different DNA pol
