Recombination, SOS response and conjugation

Question 1

what is RCR

Answer 1

rolling circle replication

Question 2

what are the methods of replication in bacteria plasmids

Answer 2

theta

RCR

Question 3

what is RCR like

Answer 3

NOT bidirectional

Does NOT use RNA primer

Question 4

where does DNA synthesis occur in RCR

Answer 4

from ‘3 OH of DNA molecule NOT RNA

Question 5

what does RCR require to finish replication

Answer 5

RecA protein

Question 6

what does RecA do

Answer 6

stimulates recombination

Question 7

what is RCR used in

Answer 7

Conjugation

Bacteriophage

Question 8

what initiates RCR

Answer 8

Ds DNA circular molecule

Starts replicating through a nick in one of the double strands in DNA, done by endonuclease enzyme

Question 9

what happens when a nick forms in dsDNA

Answer 9

can start replicating, as DNA is cut it gives you a free hydroxyl group

Question 10

where does DNA polymerase bind in RCR

Answer 10

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

Question 11

how does DNA polymerase read strand in RCR

Answer 11

free 3’ end extended by DNA polymerase 9read 5’-3’) displacing the complementary template strand which is copied in short segments by DNA polymerase

Question 12

when does replication in RCR stop

Answer 12

till at least one new copy is made

Question 13

what is the name when all different copies formed from RCR are joined

Answer 13

concatemer

Each copy contains the same set of genes in the same order

Question 14

what happens in RCR if the copy number is too low

Answer 14

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

Question 15

what releases the circular plasmid DNA in RCR

Answer 15

Recombination between homologous sequences releases circular plasmid DNA
Requires RecA protein

Question 16

what does RecA promote

Answer 16

recombination & releases free plasmid molecules – the copy number in the cell increases

Question 17

what is homologous recombination

Answer 17

breaking and rejoining of DNA molecules in a new arrangement

Question 18

what mediates bacteria homologous recombination

Answer 18

RecA protein (and others)
RecA has helicase (DNA unwinding) and DNA-dependent ATPase activities
ATP hydrolysis provides the energy for the recombination event

Question 19

what happens in a single recombination event in homologous recombination

Answer 19

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

Question 20

what happens in a double recombination event in homologous recombination

Answer 20

exchange of DNA fragments between the two circular pieces of DNA
e.g introducing mutants into the chromosome

Question 21

what is involved in a homologous recombination

Answer 21

holliday junction

Question 22

what does the holliday junction do

Answer 22

migrates along the DNA to swap more sequences

Question 23

what stops the holliday junction

Answer 23

Junction can be cut vertically or horizontally - creates different products

Question 24

what is bacterial SOS response

Answer 24

Error-prone DNA repair

A stress response system under the control of a repressor protein = LexA

Question 25

where does LexA bind

Answer 25

Binds to a 20 bp SOS or Lex box found in the promoter of LexA-controlled genes

Question 26

what induces SOS response

Answer 26

many agents which damage DNA or inhibit DNA replication | e.g. UV radiation or other mutagens

Question 27

what signal induces SOS regulon

Answer 27

single stranded DNA (ssDNA) – i.e. at stalled replication forks

Question 28

what does SOS response require

Answer 28

RecA mediated homologous recombination

Question 29

what happens when there is stalled replication

Answer 29

cell division is inhibited

Question 30

what is induced as part of the SOS response

Answer 30

long patch DNA repair mechanism is induced as part of the SOS response Uses RecA-mediated homologous recombination

Question 31

what does induction of SOS result from

Answer 31

autocatalytic cleavage of LexA (LexA is caused to cleave itself)

Question 32

what signal induces LexA self-cleavage

Answer 32

activated RecA protein

Question 33

when is RecA activated

Answer 33

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

Question 34

what happens when SOS regulon off

Answer 34

low levels of recA and unbroken LexA products

Question 35

what happens when SOS regulon on

Answer 35

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

Question 36

how is SOS regulon reset

Answer 36

remove ssDNA so no longer have stimulation of recA to LexA. Cleaved protein is replaced by whole, represses recA

Question 37

what does RecA-mediated DNA do

Answer 37

repair removes ssDNA from the cell RecA becomes unactivated LexA accumulates and SOS regulon is turned off

Question 38

what is vertical transmision

Answer 38

Plasmids are copied during cell growth and passed to both daughter cells at cell division

Question 39

what is horizontal transmission

Answer 39

Plasmids may also be transferred between cells from donor to recipient carrying plasmids from one type of bacteria into a different type of bacteria

Question 40

how is genetic information transferred horizontally

Answer 40

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

Question 41

what is the donor cell in conjugation

Answer 41

cell carrying plasmid

Question 42

what is recipient cell in conjugation

Answer 42

plasmid free cell

Question 43

what is the best studied conjugative plasmid

Answer 43

F (fertility) plasmid | Belongs to IncF group

Question 44

what do plasmids encode

Answer 44

conjugation genes Cell-to-cell contact mediated by pilus Specialised RCR replication genes

Question 45

what occurs in conjugation

Answer 45

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

Question 46

what happens in conjugation replication of cells (donor cell and recipient cell)

Answer 46

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

Question 47

what is transferred in conjugation

Answer 47

Only ssDNA is passed between cells | NOT sensitive to DNA restriction enzymes

Question 48

what do all conjugative plasmids encode genes for

Answer 48

Host cell recognition Cell-to-cell contact structures (pilus) DNA processing enzymes (nicking & unwinding)

Question 49

how will different plasmids differ in DNA transfer

Answer 49

Different plasmids will transfer DNA to different cells types Broad host range plasmids termed promiscuous

Question 50

how can plasmids without full Tra gene set be transferred

Answer 50

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)

Question 51

what is cointegration

Answer 51

Non-mobilisable plasmids can be transferred between cells Transfer as part of a conjugative plasmid following a homologous recombination event = cointegration