DNA Replication Flashcards
What is a replicon?
a unit of the genome in which DNA is replicated; each replicon contains an origin for initiation of replication
What is an origin?
a sequence of DNA at which replication is initiated
What is a plasmid?
an autonomous circular DNA that constitutes a separate replicon
How is replication bidirectional?
an origin creates 2 replication forks that move in opposite directions
What is the origin of E. coli?
oriC
What do ter sites on the bacterial genome do?
cause termination if the replication forks go too far
What regulates initiation of bacterial replication?
methylation of the bacterial origin
What does oriC contain?
11 palindromic GATC repeats that are methylated on adenine on both strands by Dam methylase
What does seqA do?
bind to hemi-methylated DNA and prevent the origin from being remethylated
What is the licensing factor of bacterial DNA replication?
dnaA
What is licensing factor?
a factor necessary for replication that is inactivated or destroyed after one round of replication
What does DnaA-ATP do?
bind to the fully methylated oriC sequences and form an oligomeric complex that melts DNA
What is DnaB?
an ATP hydrolysis-dependent 5’ to 3’ helicase
What is DnaC?
a chaperone that represses the helicase activity of DnaB
What is DnaG?
a primase that releases DnaC allowing DnaB helicase to become active and create the replication fork
What does a primase do?
synthesise an RNA chain that provides the priming end for DNA replication
What forms the replication fork?
- hexamer of DnaB
- gyrase (type II topoisomerase)
- SSBs
What do topoisomerases do?
bind to the double helix ahead of the replication fork and relieve the strain placed on the double helix as it unravels
What does DNA polymerase do?
add nucleotides to the 3’ OH end of the growing chain so that it grows in the 5’ to 3’ direction
What are the 5 types of DNA polymerase and what are their functions?
- I = major repair enzyme
- II = replication restart
- III = replicase
- IV = translesion replication
- V = translesion replication
What do DNA polymerases control?
the fidelity of replication
What is proofreading?
a mechanism for correcting errors in DNA synthesis that involves scrutiny of individual units after they have been added to the chain
What is processivity?
the tendency of a polymerase to remain bound to DNA in a single template rather than to dissociate and re-associate
How can DNA polymerases excise incorrectly paired bases?
by their 3’ to 5’ exonuclease activity
What does DNA polymerase I have?
5’ to 3’ exonuclease activity
How is fidelity of replication increased?
by proofreading (~100 to ~1000x more)
What is semi-discontinuous replication?
the mode of replication in which one new strand is synthesised continuously (leading) while the other is synthesised discontinuously (lagging)
What do all DNA polymerases require to initiate DNA synthesis?
a 3’OH priming end
How can a priming end be produced?
- RNA primer
- nick in DNA
- priming protein
What is the E. coli replicase?
DNA polymerase III - 900kD complex with a dimeric structure
What does each subunit of DNA polymerase III have?
- catalytic core
- dimerisation subunit
- processivity component
What does a clamp loader do?
place the processivity subunits on DNA, where they form a circular β clamp around DNA
How is the catalytic core of the DNA polymerase on the leading strand processive?
its clamp keeps it on the DNA
What links the 2 catalytic cores of DNA polymerase together?
2 copies of the dimering subunit tau
What does the clamp associated with the catalytic core of the DNA polymerase on the lagging strand do?
dissociate at the end of each Okazaki fragment and reassemble for the next fragment
What initiates each Okazaki fragment?
helicase DnaB interacting with the primase DnaG
What links Okazaki fragments together?
DNA ligase
What does DNA polymerase I in E. coli do?
use its unique 5’ to 3’ exonuclease activity to remove the RNA primer while simultaneously replacing it with a DNA sequence extended from the 3’ OH end of the next fragment
What do eukaryotic DNA polymerase delta and epsilon do respectively?
- delta = elongates lagging strand
- epsilon = elongates leading strand
How can bacteria avoid death when a replication fork stalls at damaged DNA?
- lesion bypass
- recombination
What do E. coli DNA polymerase IV and V do in the case of DNA lesions?
incorporate a non-complimentary base into the daughter strand
What does lesion bypass require?
error-prone DNA polymerase temporary replacement with DNA polymerase III of the replisome
What is an episome?
a segment of DNA that can exist and replicate either autonomously in the cytoplasm or as part of a chromosome, mainly found in bacteria
What is transformation?
one mode of horizontal gene transfer in bacteria, where a bacterium takes up a piece of DNA floating in its environment
How can transformation be carried out in vitro?
CaCl2 heat shock and electroporation
What is conjugation?
the process in which two bacteria come in contact and transfer genetic material mediated by F plasmid
What is a free F plasmid?
a replicon that is maintained at the level of 1 plasmid/bacterial chromosome and can be integrated into the bacterial chromosome
What happens to the F- recipient after conjugation?
it is converted to F+ (except in Hrf transfer)
Where are Tra genes encoding transfer functions found?
in the operon
Give examples of Tra gene transfer functions
- pilus synthesis and assembly
- cell pairing
- nicking at oriT
When are Hfr strains formed?
when F integrates into bacterial chromosomes
Why do most F- cells not acquire an F+ phenotype in Hfr x F- mating?
only the first part of F is transferred
What is the order of genes transferred in Hfr x F- mating?
azi-ton-lac-gal
Which genes have the highest frequency of being transferred?
those nearest the oriT
Which genes are more frequently represented in recombinants?
those that are transferred early
What are prototrophs?
wild-type strain that have minimal requirement for nutrient supplements
What are auxotrophs?
mutant strain that have lost the ability to synthesise a nutrient e.g. amino acids
How can recombinants be selected exclusively?
- counter selection against parental strains using antibiotic e.g. streptomycin
- selection using antibiotics or ability to utilise a sugar e.g. lactose
Why can’t lac- grow in the mammal medium with only lactose?
it cannot utilise lactose
How is an F’ recombinant formed?
by the improper excision of F from the bacterial chromosome
What are the 2 types of transduction?
- generalised
- specialised
When does generalised transduction take place?
during the lytic cycle
What happens in generalised transduction?
randomly sized fragments are packed into the phage and homologous recombination may occur in the recipient bacteria
What is specialised transduction?
transfer of specific portions of the bacterial genome carried out by temperate phages that have integrated their DNA into the host chromosome
When does specialised transduction take place?
when the lysogen is induced to go into lytic phase (phage induction)
What does integration and excision of phage lambda involve?
site-specific recombination
