DNA replication Flashcards
How does DNA polymerase I work and what is it?
first enzyme to be isolated by Kornberg in 1958.
is a single polypeptide chain and there are around 400 of them per cell.
it requires all 4 dNTPs, a template strand and a primer.
it isn’t self priming as it can only ad onto existing 3’-OH terminus.
Where does Pol I bind to on DNA?
binds to nicked or gapped DNA.
What direction is chain growth in polymerisation?
grows in 5’ to 3’ direction.
What are some of the activities of POL I
5’-3’ polymerase
3’-5’ exonuclease (proof reading)
5’-3’ exonuclease ( nick translation)
What is produced if a POL I is treated with proteases?
get small N terminal fragements (5’-exonuclease)
large C terminal fragments (Klenow fragment) - contains polymerase and 3-‘ exonuclease.
what is the mechanism of Klenow fragments?
the Klenow fragment requires two bound metal ions (Mg2+) held in place by aspartate residues.
it positions the 3’ end of the primer and incoming dNTP to enhance catalytic reaction.
its shape is complementary to the correctly form base pair leads to specificity as the wrong paring wont be able to fit through the fragment correctly as wrong pairings are the wrong shape and size.
retains polymerase and 3’ to 5’ exonuclease to remove 3’ overhangs or fill in 5’ overhangs to form blunt ends.
How does the Klenow fragment know which nucleotide to add?
formation of correct H bonds.
shape as incorrect base pairs wont fit into the helix.
What is the main eukaryotic DNA replication enzyme?
DNA POL III
Features of POL III?
only 10 per cell. has a high rate of polymerisation around 1600 nucleotides per second.
is highly pocessive and is a complex multimeric enzyme.
polymerases in 5’-3’ and 3’-5’ exonuclease activity for proof reading.
it does not contain 5’-3’ exonuclease activity for nick translation.
How can we determine which enzymes (proteins) are important in DNA replication?
use temperature sensitive mutants. divided into two broad categories
quick stop mutants that immediately stop DNA replication
slow stop mutants that allow replication to finish but cant start another round.
Why are there so many enzymes in DNA replicaiton?
DNA strands are antiparralel
all polymerases are 5’ to 3’ and are not self priming so need a primer
DNA strands are plectonemically coiled and need to be physically separated for semi conservative replication (e.g with DNA helicase)
How is the lagging strand of the 3’ to 5’ direction synthesised?
made up in pieces called Okazaki fragments. they are then joined together via DNA ligase. (requires ATP or NAD to join the nicks)
why does Uracil appear in DNA?
gets incorporated in place of T but is non offensive as has the same base paring as T. Can also appear from the spontaneous deamination of C but this is offensive as makes a GU pairing in place of GC which causes mutation to AT in the next round of replication.
how is Uracil removed form DNA? why did Okazaki think both strands were discontinuous?
gets removed with uracil-N-Glycosylase and it removes U opposite G but does not discriminate against U opposite A. during replication temporary nicks are created making pseudo Okazaki fragments on the leading strands.
How is proof reading done?
strand extension at the 3’ end only proceeds if the correct base has been added if not then the 3’exonuclease activity of pol III (dnaQ) removes it.
dnaQ - mutants accumate errors.
What syntheses primers and why are they needed?
synthesised by primase (dnaG). required to allow DNA polymerases to extend onto the 3’-OH end.
What is primase?
an RNA polymerase that is self priming and works in 5’ to 3’.
has no self editing functions such as proof reading and tend to be around 5- 10 nucleotides long.
activity of the enzyme is increased by the presence of a helicase.
How can we tell that primase is an RNA polymerase?
When Okazaki fragments are treated with RNase and then centrifuged get an intermediate band between RNA and DNA showing that the fragments are made of DNA and RNA (from the primers)
before Okazaki fragements are joined together via DNA ligase what occurs to the RNA primers?
POL I removes the primers and RNA and then DNA ligase cans seal the gap. (Nick translation)
What gene produces DNA helicases
dnaB