Recombination, SOS response and conjugation Flashcards
what is RCR
rolling circle replication
what are the methods of replication in bacteria plasmids
theta
RCR
what is RCR like
NOT bidirectional
Does NOT use RNA primer
where does DNA synthesis occur in RCR
from ‘3 OH of DNA molecule NOT RNA
what does RCR require to finish replication
RecA protein
what does RecA do
stimulates recombination
what is RCR used in
Conjugation
Bacteriophage
what initiates RCR
Ds DNA circular molecule
Starts replicating through a nick in one of the double strands in DNA, done by endonuclease enzyme
what happens when a nick forms in dsDNA
can start replicating, as DNA is cut it gives you a free hydroxyl group
where does DNA polymerase bind in RCR
DNA polymerase recognizes 3’ hydroxyl group, associates with that end then starts adding on new bases, causing plasmid to roll out, form new DNA
Form long piece of DNA with repeated copies on it
how does DNA polymerase read strand in RCR
free 3’ end extended by DNA polymerase 9read 5’-3’) displacing the complementary template strand which is copied in short segments by DNA polymerase
when does replication in RCR stop
till at least one new copy is made
what is the name when all different copies formed from RCR are joined
concatemer
Each copy contains the same set of genes in the same order
what happens in RCR if the copy number is too low
Rep is released & no termination occurs
New “+ strand” is not circularised Linear concatamers are created and lagging strand synthesis converts these to dsDNA
Circular plasmids are created by recombination
what releases the circular plasmid DNA in RCR
Recombination between homologous sequences releases circular plasmid DNA
Requires RecA protein
what does RecA promote
recombination & releases free plasmid molecules – the copy number in the cell increases
what is homologous recombination
breaking and rejoining of DNA molecules in a new arrangement
what mediates bacteria homologous recombination
RecA protein (and others) RecA has helicase (DNA unwinding) and DNA-dependent ATPase activities ATP hydrolysis provides the energy for the recombination event
what happens in a single recombination event in homologous recombination
can fuse two things together –two whole plasmids
Can integrate a plasmid into a chromosome or can integrate a plasmid into another plasmid done through recombination
what happens in a double recombination event in homologous recombination
exchange of DNA fragments between the two circular pieces of DNA
e.g introducing mutants into the chromosome
what is involved in a homologous recombination
holliday junction
what does the holliday junction do
migrates along the DNA to swap more sequences
what stops the holliday junction
Junction can be cut vertically or horizontally - creates different products
what is bacterial SOS response
Error-prone DNA repair
A stress response system under the control of a repressor protein = LexA
where does LexA bind
Binds to a 20 bp SOS or Lex box found in the promoter of LexA-controlled genes
what induces SOS response
many agents which damage DNA or inhibit DNA replication
e.g. UV radiation or other mutagens
what signal induces SOS regulon
single stranded DNA (ssDNA) – i.e. at stalled replication forks
what does SOS response require
RecA mediated homologous recombination
what happens when there is stalled replication
cell division is inhibited
what is induced as part of the SOS response
long patch DNA repair mechanism is induced as part of the SOS response
Uses RecA-mediated homologous recombination
what does induction of SOS result from
autocatalytic cleavage of LexA (LexA is caused to cleave itself)
what signal induces LexA self-cleavage
activated RecA protein
when is RecA activated
ssDNA appears in the cell (i.e. stalled replication forks)
Basal level of RecA = 1200 molecules per cell
After induction = 60,000 molecules per cell
what happens when SOS regulon off
low levels of recA and unbroken LexA products
what happens when SOS regulon on
recA detects single stranded DNA, detect damage and stalled replication forks, stimulates LexA to cleave itself. Role is to get rid of ssDNA, needs to carry on with replication, however will produce errors in the process
how is SOS regulon reset
remove ssDNA so no longer have stimulation of recA to LexA. Cleaved protein is replaced by whole, represses recA
what does RecA-mediated DNA do
repair removes ssDNA from the cell
RecA becomes unactivated
LexA accumulates and SOS regulon is turned off
what is vertical transmision
Plasmids are copied during cell growth and passed to both daughter cells at cell division
what is horizontal transmission
Plasmids may also be transferred between cells from donor to recipient
carrying plasmids from one type of bacteria into a different type of bacteria
how is genetic information transferred horizontally
Pilus reaches out and grabs another bacterium and pulls it into it, pass genetic material – copies plasmid into other cell its grabbed, both contain the plasmid
what is the donor cell in conjugation
cell carrying plasmid
what is recipient cell in conjugation
plasmid free cell
what is the best studied conjugative plasmid
F (fertility) plasmid
Belongs to IncF group
what do plasmids encode
conjugation genes
Cell-to-cell contact mediated by pilus
Specialised RCR replication genes
what occurs in conjugation
Contact is made between donor cell and plasmid free recipient by pilus
Pilus then retracts pulling cells together
Cell walls fuse
DNA does NOT pass down an extended tube
what happens in conjugation replication of cells (donor cell and recipient cell)
Nick occurs at origin of transfer (oriT) by TraY/I proteins
These proteins initiate DNA replication
ssDNA is transferred to recipient with 5’ end leading
New second strand is made in both donor and recipient
Cells actively separate at the end of DNA transfer
what is transferred in conjugation
Only ssDNA is passed between cells
NOT sensitive to DNA restriction enzymes
what do all conjugative plasmids encode genes for
Host cell recognition
Cell-to-cell contact structures (pilus)
DNA processing enzymes (nicking & unwinding)
how will different plasmids differ in DNA transfer
Different plasmids will transfer DNA to different cells types
Broad host range plasmids termed promiscuous
how can plasmids without full Tra gene set be transferred
Must be in same cell as a conjugative plasmid-must be compatible
bom site (basis of mobility)
Equivalent of oriT site
bom site may be nicked by enzymes from conjugative plasmid OR by own mobilisation genes (mob genes)
what is cointegration
Non-mobilisable plasmids can be transferred between cells
Transfer as part of a conjugative plasmid following a homologous recombination event
= cointegration
