Exam 3: DNA Replication Flashcards
Explain Meselson and Stahl’s experiment
-N14 and N15 centrifuged separately. Lighter N14 always at the top of tube and heavier N15 at the bottom of tube.
-N15 E.coli grown in medium containing N14.
-N centrifuged and found to be in the middle of N14 and N15 bands which proved that the DNA had replicated semiconservatively and was half of each.
Prokaryotic DNA replication begins at the
ORI (origin of replication)
Replication fork
Site of replication where helix is unwound
Replication is
bidirectional (it starts in one place and goes in two directions); therefore, there are two replication forks
Replicon
Length of DNA replicated
DNA polymerase 1
Enzyme catalyzes directional DNA synthesis: chain elongation occurs 5’ to 3’
Requires DNA template, a primer, and all four deoxyribonucleoside triphosphates (dNTPs)
Exonuclease Activity
3’-5’
All three possess 3’ to 5’ exonuclease activity; proofread newly synthesized DNA, remove/replace incorrect nucleotide.
Dramatically increases the fidelity of the replicating enzyme.
5’-3’
Only DNA polymerase 1
Excises primers– fills in gaps left behind
Which DNA polymerase have the initiation of chain synthesis?
None
What DNA polymerase has 5’ to 3’ exonuclease activity?
DNA polymerase 1
Seven key issues that must be resolved during DNA replication
- Unwinding of the helix
DNAa initiator proteins
SSB proteins
Helicase
- Reduce increased coiling generated during unwinding
Topoisomerase/DNA Gyrase
- Synthesis of primer for initiation
Primase
- Discontinuous synthesis of second strand
DNA Polymerase 3
- Removal of the RNA primers
DNA Polymerase 1
- Going of gap-filling DNA to adjacent strand
DNA Ligase
- Proofreading
DNA polymerase exonuclease activity of 3ʹ–5ʹ allows for excise of nucleotides
Components needed to synthesis molecules
Template
Substrates= dNTP (deoxynucleoside triphosphate)
Proteins/enzymes to coordinate assembly of substrate
DnaA
-Initiator protein encoded by DnaA gene
-Binds to ORI causing conformation to change
-Causes helix to destabilize and open up
-Exposes ssDNA
DNA Helicase
-Made of DnaB polypeptides
– Subsequently recruits holoenzyme to bind
replication fork and initiate replication
– Helicases require energy supplied by hydrolysis of ATP denatures hydrogen bonds and stabilizes double helix
SSBPs
-Single-stranded binding proteins
-Stabilize the open conformation of helix. Bind specifically to single strands of DNA.
How does DNA replication avoid supercoiling?
DNA gyrase
–Enzyme relieves coiled tension from unwinding of helix (DNA supercoiling) by making single- or double-stranded breaks
–Driven by energy released during ATP hydrolysis