Replication Flashcards
What are inferred types of models for DNA Replication?
Conservative: original model is conserved when synthesizing a completely new double stranded DNA
Semiconservative: each parental “old” strand serves as a template for synthesis of “new” daughter DNA strand
Dispersive: original molecule is replicated and each strand in the daughter molecule is a combination of synthesized and parent DNA
What is the Meselson and Stahl experiment?
Two groups of cells of E. coli grown, one with nitrogenous bases of N15 isotope and the other with N14 isotope. N15 E. coli is added to N14 medium. DNA is extracted and spun via density gradient centrifugation.
How does the Meselson and Stahl Experiment determine the correct model of DNA replication?
After the first replication, density gradient medium reveals a single band of N14/N15 (ruling out conservative), after second replication medium reveals two bands for N14 and N15 (ruling out dispersive)
What is the bidirectional progression of replication of E. coli?
- Replisome binds to DNA and initiates synthesis at replication forks
- Replication forks continue synthesis in opposite directions
- Replication forks hit terminus of replication and collide, releasing two chromosome copies
How is the replication of E. coli’s chromosome discovered?
E. coli was fed radioactive precursor (thymine) and was exposed to film to demonstrate oriC (initiation site) and ter (termination. site) as well as the replication bubble.
What is a consensus sequence
a common nucleotide sequence or amino acid sequence found in highly conserved regions of DNA or RNA or proteins
What are the regions on the E. coli oriC?
- AT rich regions
- Sites for DnaA binding
- Methylation sites
- Sites for FIS and IHF proteins
What is dnaA?
Origin binding protein: Protein that binds origin of replication to open double helix
What is dnaC?
Helicase loader: Loads helicase at origin
What is dnaB?
Helicase: Protein that unwinds double helix at replication fork
What is ssb?
Single strand binding protein: prevents single strands from annealing
What is HU (heat unstable protein)
Histone-like protein involved in bending and folding and packaging DNA in bacteria
What is dnaG?
Primase: produces RNA primer
What is dnaN?
holds DNA Polymerase III onto DNA
What are the steps of initiation of replication?
- dnaA binds to high affinity dna A binding sites, which is used as an anchor to cooperatively bind ATP-dnaA complexes to the low affinity AT-rich region
- Topological stress breaks H-bonds and unwinds AT-rich region with the helps of HU, which provides a higher affinity to ATP-dnaA molecules
- ssb proteins stabilize single stranded DNA which prevents helix formation
- 6 monomers of dnaC recruits 6 monomers of dnaB onto each single strand
- dnaB monomers form a hexameric ring around each strand and dnaC monomers dissociate
- After helixase moves outward a bit, primase is loaded behind each helicase
- Sliding clamp is loaded between helicase and DNA polymerase III, while ATP-dnaA hydrolyzes to ADP-dnaA which promotes dissociation
- Primase lays down RNA primers that has 3’ OH ends that will be recognized by DNA polymerase III for DNA synthesis
