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Applied Genetics > Recombination, SOS response and conjugation > Flashcards

Recombination, SOS response and conjugation Flashcards

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Biology 101 flashcards
1
Q

what is RCR

A

rolling circle replication

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2
Q

what are the methods of replication in bacteria plasmids

A

theta

RCR

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3
Q

what is RCR like

A

NOT bidirectional

Does NOT use RNA primer

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4
Q

where does DNA synthesis occur in RCR

A

from ‘3 OH of DNA molecule NOT RNA

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5
Q

what does RCR require to finish replication

A

RecA protein

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6
Q

what does RecA do

A

stimulates recombination

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7
Q

what is RCR used in

A

Conjugation

Bacteriophage

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8
Q

what initiates RCR

A

Ds DNA circular molecule

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

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9
Q

what happens when a nick forms in dsDNA

A

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

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10
Q

where does DNA polymerase bind in RCR

A

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

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11
Q

how does DNA polymerase read strand in RCR

A

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

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12
Q

when does replication in RCR stop

A

till at least one new copy is made

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13
Q

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

A

concatemer

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

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14
Q

what happens in RCR if the copy number is too low

A

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

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15
Q

what releases the circular plasmid DNA in RCR

A

Recombination between homologous sequences releases circular plasmid DNA
Requires RecA protein

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16
Q

what does RecA promote

A

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

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17
Q

what is homologous recombination

A

breaking and rejoining of DNA molecules in a new arrangement

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18
Q

what mediates bacteria homologous recombination

A 
RecA protein (and others)
RecA has helicase (DNA unwinding) and DNA-dependent ATPase activities
ATP hydrolysis provides the energy for the recombination event
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19
Q

what happens in a single recombination event in homologous recombination

A

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

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20
Q

what happens in a double recombination event in homologous recombination

A

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

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21
Q

what is involved in a homologous recombination

A

holliday junction

22
Q

what does the holliday junction do

A

migrates along the DNA to swap more sequences

23
Q

what stops the holliday junction

A

Junction can be cut vertically or horizontally - creates different products

24
Q

what is bacterial SOS response

A

Error-prone DNA repair

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

25
Q

where does LexA bind

A

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

26
Q

what induces SOS response

A

many agents which damage DNA or inhibit DNA replication

e.g. UV radiation or other mutagens

27
Q

what signal induces SOS regulon

A

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

28
Q

what does SOS response require

A

RecA mediated homologous recombination

29
Q

what happens when there is stalled replication

A

cell division is inhibited

30
Q

what is induced as part of the SOS response

A

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

31
Q

what does induction of SOS result from

A

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

32
Q

what signal induces LexA self-cleavage

A

activated RecA protein

33
Q

when is RecA activated

A

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

34
Q

what happens when SOS regulon off

A

low levels of recA and unbroken LexA products

35
Q

what happens when SOS regulon on

A

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

36
Q

how is SOS regulon reset

A

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

37
Q

what does RecA-mediated DNA do

A

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

38
Q

what is vertical transmision

A

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

39
Q

what is horizontal transmission

A

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

40
Q

how is genetic information transferred horizontally

A

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

41
Q

what is the donor cell in conjugation

A

cell carrying plasmid

42
Q

what is recipient cell in conjugation

A

plasmid free cell

43
Q

what is the best studied conjugative plasmid

A

F (fertility) plasmid

Belongs to IncF group

44
Q

what do plasmids encode

A

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

45
Q

what occurs in conjugation

A

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

46
Q

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

A

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

47
Q

what is transferred in conjugation

A

Only ssDNA is passed between cells

NOT sensitive to DNA restriction enzymes

48
Q

what do all conjugative plasmids encode genes for

A

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

49
Q

how will different plasmids differ in DNA transfer

A

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

50
Q

how can plasmids without full Tra gene set be transferred

A

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)

51
Q

what is cointegration

A

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

Applied Genetics (20 decks)

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