DNA Replication and Transfer Flashcards
Define replicon
Unit of genome where DNA is replicated = each containing an origin for replication initiation
Define origin
Sequence of DNA where replication is initiated
Define plasmid
Autonomous circular DNA that makes up a separate replicon
Define single-copy replication
Replicates once every time bacterial chromosome replicates
Define multi-copy replication
Greater number of copies per individual bacterial cell
Describe the replication fork
Replication fork = initiated at origin and moves along DNA
Bidirection
When is replication bidirectional?
When 1 origin creates 2 replication forks that move in opposite directions
Describe a bacterial genome
Usually a single circular replicon
Replicate bidirectionally from single origin
How do replica chromosomes split into daughter cells?
Decatenated
What does oriC contain?
11 palendromic GATC reapeats
Methylated on ADENINE on both strands
What enzyme methylates the adenine in palindromic oriC?
Dam methylase
What type of DNA is produced from replication?
Hemimethylated DNA = cannot initiate replication
What does the origin need to initiate replication?
Only fully methylated origins can initiate replication
How long before new origin is remethylated?
13 minute delay until methylated by Dam methylase
What is required for delaying rereplication?
SeqA = binds to hemimethylated DNA
Prevents origin being remethylated
For initiation to occur what is needed?
There must be membrane or cell wall synthesis
What is the effect of penicillin?
Inhibits cell wall synthesis = blocking replication initiation
What is the bacteria licensing factor?
DnaA = inactivated or destroyed after one round of replication
What does DnaA need to perform its role?
ATP
What is the role of DnaA-ATP?
Melts DNA by binding short repeated sequence that is fully methylated
What is the role of DnaB?
ATP hydrolysis-dependent 5’to3’ helicase
What is the role of DnaC?
Chaperone = represses the helicase activity of DnaB
What is the DnaB/C complex called and where does it bind?
Prepriming complex that binds origin
Helicase is inactive until DnaC leaves
What is the role of DnaG?
Primase = binds to helicase complex
What does a primase do?
Synthesizes an RNA chain
Providing priming end for DNA replication
What forms the replication fork?
Hexamer of DnaB = helicase
What is gyrase and where does it bind?
Type II topoisomerase
Binds ahead of replication fork = relieves strain placed in the double helix as it unravels
How does DNAp carry out proofreading?
3’ to 5’ exonuclease that excises incorrectly paired bases
What is processitivity?
Tendancy to remain on a single template rather than dissociate and re-associate
What has a 5’ to 3’ exonuclease?
DNAp I
What does DNAp need to initiate DNA synthesis?
RNA primer = synthesized by primase
What sub-complexes does DNAp holoenzyme have?
Each monomeric unit has
Catalytic core, dimerization subunit and processivity component
What is one catalytic core associated with?
One core = associated with one template strand
What is the role of the clamp loader?
Places processivity subunit on DNA
These two subunits for a beta clamp around DNA
What is the role of dimerizing subunit t?
Links the two catalytic cores together
What happens to the clamp on the leading and lagging strand?
Leading strans is processive because its clamp keeps it on DNA
Clamp on lagging strand dissociates at end of each Okazaki fragment and reassembles for next fragment
How are each Okazaki fragments initiated?
Helicase DnaB is responsible for interacting with primase DnaG to initiate
What is the role of DNAp I?
Removes RNA primer = using 5’ to 3’ exonuclease
Replaces it with DNA
What happens when a replication fork reaches damaged DNA and how can bacteria avoid death?
Replication fork stalls and may collapse
Lesion bypass or homologous recombination
How does lesion bypass work?
Error-prone DNAp IV = replicates past the lesion allowing replication to continue
This may produce mutations in the sequence
What are the 3 gene transfer processes in prok?
Transformation = naked DNA
Conjugation = bacteria-mediated
Transduction = phage-mediated
What is an epsiome?
Segment of DNA = can exist and replicate either autonomously in the cytoplasm or as part of chromosome
What are two methods of gene transfer via transformation?
CaCl2 then heatshock
Electroporation
What is conjugation?
Process where genetic material is transferred by F plasmid
F = fertility factor
What is the free F factor?
Replicon maintained at the level of 1 plasmid
It can be integrated into bacterial chromosome
What are tra genes and where are they located?
Encoding transfer function = pilus synthesis and assembly, cell pairing nicking at oriT
Located in an operon
What is the mechanism of conjugation?
Rolling circle mechanism
Nick at origin by TraY/I
TraY/I unwinds DNA
Elongation of growing strand displaces old strand
After one revolution displaced strand reaches unit length
TraI bound single strand enters recipient
What strains are made when F integrates into bacterial chromosome?
High-frequency of recombination strains
Plasmid must recombine with bacterial chromosome to become a Hfr cell
What happens in Hfr x F- mating?
F- cells almost never acquire F+ phenotype
Because only the first part of F is transferred
What is an exconjugant?
Each of the cells that separates after conjugation has taken place
What are prototrophs vs auzotrophs?
Prototrophs
Wild-type strain = has minimal requirement for nutrient supplements
Auxotrophs
Mutant strain = lost ability to synthesize nutrients (amino acids)
