Textbook 1.2 Flashcards
Pg. 394-398, 382-387, 460-461, 465-470, 480-482, 512-526
DNA Gyrase
Type of topoisomerase that relieves mechanical strain that builds up during replication in E. coli (super coiling). Cleaves DNA duplex in two and passes one of the strands through. Eukaryotes have similar enzymes which have same function.
For DNA polymerase to work…. must be present
DNA and all four deoxyribonucleoside triphosphates dTTP, dATP, dGTP, dCTP
All DNA polymerases CANNOT…and thus require …
- initiate the formation of a new DNA strand—can only add nucleotides onto the 3’ OH terminus of an existing strand
- a template strand to copy and a primer to provide the OH end
What is the major enzyme responsible for DNA replication…
DNA Polymerase III, even though there is far fewer copies of the enzyme than Polymerase I which was discovered first.
Replication is said to be…
semi-discontinuous
Which enzyme links Okazaki fragments together
DNA ligase
Which enzyme initiates DNA replication
An RNA polymerase called RNA primase which constructs a short primer of RNA
What happens to the RNA primers at the end of replication?
They are removed and replaced with DNA. The strands are then sealed by DNA ligase.
What is a possible reason for an RNA primer being used?
Potentially more mistakes made during initiation than elongation so a short, removable RNA primer avoids inclusion of mismatched bases.
Helicase
Unwinding enzyme that uses energy from hydrolysis of ATP to move along strand. Forms a ring like structure around strand.
SSB Proteins
Single-stranded DNA-binding proteins: bind selectively to single stranded DNA to keep it extended and prevent it from rewinding or getting damaged.
Primosome
Transient association between primase and helicase in bacteria during replication.
Describe the polymerase complex during replication
Both the leading and lagging strand have separate DNA polymerases which are adding onto the 3’ OH terminus of the leading and lagging strand. The DNA polymerase on the leading strand is synthesized continuously while the protein on the lagging strand synthesizes in chunks then releases template to move along to the next fragment. It is hitching a ride on the other polymerase. They are part of one complex even though there are two polymerases because the DNA of the lagging strand template is looped back on itself. They are moving towards opposite ends of their respective templates but ultimately still in same direction.
DNA polymerase III holoenzyme
replication machine. Includes:
- DNA polymerase III
- beta clamp (keeps polymerase associated w template)
- clamp loader (holds clamp open for DNA to squeeze into ring of beta clamp)
Difference between beta clamp and DNA polymerase III association on the leading and lagging strand
Polymerase on the leading strand remains tethered to the beta clamp for the entire duration of the replication while polymerase on the lagging strand disengages from the beta clamp after completing an Okazaki fragment and is cycled to a new beta clamp on an RNA primer-DNA template junction closer to replication fork.
Exonucleases can be divided into…
5’ to 3’ and 3’ to 5’ depending on the direction of the degradation of the DNA strand
Why is DNA polymerase I almost like three enzymes in one?
It has polymerizing capabilities but also 3’ -> 5’ and 5’ -> 3’ exonuclease capabilities. These activities are found in three different domains of the polypeptides. The 5 to 3 exonuclease is not RNA/DNA specific unlike most other exonucleases and can degrade both.
Which enzyme removes the RNA primers that initiate okazaki fragments?
5’ to 3 ‘ exonuclease activity of DNA polymerase I. It also fills the gaps as it removes the primers.
Which enzyme seals okazaki fragments together
DNA ligase
How does DNA polymerase ensure the right nucleotides are being added?
Bond angles and geometry of base pairs. If the nucleotide has the proper fit the enzyme will undergo a conformational change to add onto the growing chain. If it does not have proper geometry enzyme cannot achieve conformation required for catalysis.
What happens if the wrong nucleotide is added?
The frayed end of the 3’ strand is fed into the 3’ to 5’ active nuclease site (at wrist) where the incorrect base is removed. This removes 99 out of every 100 mismatched bases.
Fidelity of DNA replication relies on…
- accurate selection of nucleotides
- immediate proofreading
- post-replicative mismatch repair