Recombination Flashcards
simple def. of recombination
2 DNA molecules broken at same place and rejoined in new combinations
2 purposes of recombination
1) evolution
2)facilitation of DNA replication (eg. DNA repair)
type of genetic evolution
A) change gene order (deletions, inversions)
B) move genes to different replicons
i) formation of recombinants after conjugation between hfr strain and F- recipient
ii) formation of recombinants after gen/spec transduction
iii)integration of plasmid DNA into chr (Hfr formation)
iv) integration of phage DNA into chr (ex. lysogeny)
facilitation of DNA replication/repair: Restarting collapsed DNA replication forks likely selective advantage that led to evolution of ______
- Bacterial chromosomes have _____ so nicks in template potentially fatal
-homologous recombination
-only one ori
types of recombination: ______: more rare; requires special proteins that promote recombination between _____
-______: occurs more often; between any 2 same / similar DNA sequences (~ ____ bases minimum/____ bp more efficient); both produce DNA breakage & rejoining/ crossovers
-Site-specific
-short specific sequences
-Homologous
-20
-500
site-specific recombination involves which proteins?
Transposon transposase, Phage integrase,
homologous recombination involves which proteins?
Host Cell Machinery, (acts on Transposons, ISs, 2 copies of similar DNA in the cell, etc.)
discovery of recombination: Hershey & Rotman, 1940. Lab: coinfected E. coli with ____ and _____ T2 phage each at MOI = 5 –> 98% parental, 2% equal numbers of r-h- & r+h+
- r-h- and r+h+ breed true -> genotypes obtained due to _____
- Showed ____ are suitable objects for genetic analysis
- r-h+
-r+h-
-recombination
-phages
Historical Models of Recombination: 2 models put forth to explain generation
of recombinant progeny:
Copy choice (def.)
Breakage & reunion (def.)
-recombinants are generated during replication by copying first one parental sequence and then the other
-recombinant sequence is generated by association of DNA fragments from different parental lines
Meselson & Weigle, 1962 -> test ____ model in bacteriophage lambda:
- Phage DNA replicated in media with ____ had heavier density than phage
DNA replicated with _____
- coinfect E. coli with heavy DNA ____
and light DNA ____
- measure density of ____
- recombinant progeny range in density from fully heavy to fully light = each recombinant particle contains _____
-therefore, recombination involves _____ of genetic material. Experiments indicated parental DNA was breaking and recombining, giving ______ and that _____ was not happening, or else there would be one density of copy
choice DNA (in N14 media).
-breakage & reunion
-N15
-normal N14
-A+R- lambda phage
-A-R+ lambda phage
-recombinant progeny
-genetic material from both parents
-physical exchange
-different density recombinants
-copy choice
requirements for homologous recombination (4)
1) Identical/Very Similar Sequences in Crossover Region = ensure recombination is site-specific 9no genetic material lost)
2) Complementary Base Pairing Between Double-Stranded DNA Molecules
3) Recombination Enzymes (nucleases, ligases)
4) Heteroduplex Formation
synapse (def.) + intial formation rquires mechanism for complemntary pairing between 2 ds helices without _____
-point where 2 DNA molecules held together by complementary base pairing between strands
-strand separation
heteroduplex (def.)
- regions of complementary base pairing in synapse, strands in this region are from different DNA molecules ( all strands of 2 DNA molecules involved)
Holliday Double-Strand Invasion Model - Robin Holliday, 1964: 2 single-stranded breaks at same place in 2 DNA molecules; free ends cross over each other & pair with complementary sequence in other DNA molecule to form ______. ends ligated to each other to form cruciform structure aka ________. Holliday junctions can isomerize between ______. Holliday junction cut and religated to be _____. Occurrence of recombination dependent on _____ of Holliday junction at resolution
-2 heteroduplexes
-holliday junctions
-2 different forms
-resolved
-configuration
BRANCH MIGRATION: Holliday junctions (synapses) can move ____ the length of the DNA. Specific proteins are required to ______ rapidly; this can _____ the size of the heteroduplex region. BUT, there is no evidence for a mechanism that ensures strand cutting at ______. Most recombination models include ______; but differ in earlier stages
-up & down
-break & reform H-bonds
-increase
-exactly same place in both molecules
- Holliday junctions & branch migration
Single-Strand Invasion - Meselson and Radding: strand of one of 2 DNA molecules cut at ____. exposed end invades another ______ until finds complementary sequence; displaces one strand; _____ fills in gap left by invading strand; displaced strand _____; remaining end ligated to newly synthesized DNA on other DNA molecule -. Heteroduplex originally forms in only 1
DNA molecule; branch migration causes ______
-random
-double-stranded DNA
-DNA pol
-degraded
-heteroduplex formation in the other DNA molecule
Double-Strand Break Repair: experimental evidence supports dsDNA breakage in some recombination
situations, despite _____; both strands of 1 DNA molecule broken (5’ ends digested by _____ (gap_s, one 3’ tail invades other double-stranded DNA molecule until finds complementary sequence)
-_____ extends tail along complementary sequence, displacing other strand
-when reaches 5’ end of invading strand, sealed by ligase, other free 3’ end used as primer by _____
to fill in gap, using displaced strand as______= 2 Holliday structures formed
-assumed lethality
-exonuclease
-DNA pol
- DNA Pol
- template DNA
How do we know that recombination & replication are connected?
1) Stable DNA Replication - Normally, initiation of DNA replication does NOT require _____ functions but DOES require protein synthesis
- After severe DNA damage, another type of DNA replication initiation occurs -> ______ (FORM OF REPAIR)
* does not require protein synthesis
* uses ______ to initiate replication via recombination
* uses ___ proteins & ____ to reload DnaB helicase through ____
*initiation of DNA replication with Pri proteins is required for the ______ that rescues collapsed replication fork
-recombination
-stable DNA replication
-dsDNA breaks
-Pri
-DnaT
-DnaC
-homologous recombination event
Simple overview of homologous recombination: Single-stranded DNA end generated (_____ or _____/_____). RecA coats ___ & stimulates synapse formation between _____ in other DNA molecules (Holliday junction) . ____ proteins catalyze resolution event
-RecBCD
-RecQJ/RecFOR
-ssDNA
- ssDNA and homologous sequences
-RuvABC/RecG
RecBCD is four things?
ssDNA endonuclease
ssDNA exonuclease
DNA helicase (2x -> opposite polarity)
DNA-dependent ATPase
RecBCD recognizes & loads on ______ and _____ unwinds DNA creating loop that is cut into small pieces by _______; it continues til ____ reached. Then ____ inhibited, _____ active and _____ stimulated by RecD; this generates free ______
-flush dsDNA ends
-RecD
-RecB 3’ -> 5’ exonuclease
-X site
-RecB 3’ -> 5’ exonuclease
-RecB helicase
-RecB 5’ -> 3’
-3’-OH
- RecC recognizes _____
- Rec signals ___ to stop
- When stopped, RecD signals ____ to cut DNA
- RecB cuts where it is and unwinding DNA loading ____
-chi
-RecD
-RecB
-RecA
RecBCD -> RecA loading: ____ stimulates cooperative binding of RecA to ____. ____ inhibits RecA binding until _____ reached & ____ inactivated. Cooperative binding important since ___ binds DNA better than RecA. More RecA binds cooperatively to form helical filament (_____ substrate). RecBC loading of RecA ensures RecA only bound where _____
-RecBC
-3’ ssDNA
-RecD
-X site
-RecD
-SSB
-recombination
-recombination will occur.
RecBCD -> X sites- X site interacts with ____ which acts to inhibit RecD, which then inhibits ______. RecD also stimulates ____ endonuclease to cut ____ across from x. RecB then uses _____ to degrade non-x strand. ___ coats x strand ssDNA with RecA
-RecC
-RecB 3’ -> 5’exonuclease
-RecB
-ssDNA
- 5’ -> 3’ exonuclease
-RecB
recD- mutants: are ____, and fail to degrade transformed _____. Proficient for _____. does not require _____
-viable
-linear DNA
-recombination
-x sites
why have x sites at all?
1) defense against foreign DNA-> immunity
2) control direction of DNA replication following ds break
evidence for x sites role in immunity
- x sites over-represented in E. coli genome: 1:4.5 kb
- By chance alone: 1:65 kb
- foreign DNA will be degraded by RecBCD
phages overcome bacterial immunity by protecting _____ and/or making proteins that inhibit ____
-dsDNA ends
-RecBCD (ie. lambda Gam)
x like sites found in many ______; Different sequences; Work with different enzymes, similar to _____
-other microbes
-RecBCD
how x sites control direction of DNA replication following ds break?
-Two ends where RecBCD could load on dsDNA break
* x sites only work in one direction( polar); RecBCD will only generate 3’ end for recombination when encounters x site in right orientation
* Polar x sites enriched in one orientation
* Causes new replication forks initiated by RecBCD action to be in same direction as those initiated from oriC
RecF Pathway is another way to generate _____ for recombination
ssDNA
RecF pathway used to initiate recombination at ssDNA gaps generated during _______ & ______; important for restarting ______
-DNA repair
-movement of replication fork past lesion in lagging strand
-replication
RecF pathway.____ (helicase) opens DNA then___ (exonuclease) creates ssDNA , ___ binds to ssDNA, _____ then load RecA then ____ causes recombination
-RecQ
-RecJ
-SSB
-RecFor
-RecBCD
RecA nucleoprotein filament formed that is _____ on ssDNA with help of ______. Must scan genome to find complementary DNA & catalyze _____!!
Very efficient -> incoming ssDNA sequence recombines with _____ in cell ~ 100% of the time!!! It’s reading DNA sequence without _____
-helical & extended
-RecFOR or RecBCD.
-strand invasion
-complementary DNA
-pulling apart the strands
RecA nucleoprotein filament scans DNA via _____. Base pairs with complementary sequence without _____.
Possible models?
① ________
②__________
-major groove
-disrupting helix
-displacement of one strand & formation of a D loop
-formation of triple stranded structure
How RecA coated ssDNA finds complementary DNA and invades is still mysterious.
A RecA coated ssDNA can ACTIVATE ______ & allow invasion of a complementary ssDNA
-Called _______”; activated helix may be transiently extended and strands partially separated . After invasion, RecA can extend nucleofilament into neighbouring dsDNA -> _____
-another (non complementary) dsDNA
-“TRANS-ACTIVATION
-SPOOLING
Holliday junctions are special junctions created by _______ into dsDNA. Can migrate along DNA (_____). Catalyzed by specialized enzymes. Create heteroduplexes -> (def.) Holliday junctions can ISOMERIZE -> (def.) Requires no energy, happens quickly, no ____ broken! Configuration of strands upon resolution determines how _______
-RecA mediated invasion of ssDNA
-branch migration
-complementary base pairing between 2 DNAs from different molecules
-rearrangement that. changes relationship of strands to one another
-H-bonds
-recombination between markers occurs
protein involved in migration and resolution of Holliday junctions: RuvA (def.)
-Flower-like tetramer that binds crossed DNA on both sides in flat configuration amenable to cutting
-Single strands form non H-bonded square in middle of tetramer
protein involved in migration and resolution of Holliday junctions: RuvB
- Hexameric ring that encircles one arm of DNA
- Cleaves ATP to pump DNA through, moving Holliday junction
protein involved in migration and resolution of Holliday junctions: RuvC
-resolves Holliday junctions -> cuts at 2 ssDNAs at specific sequence 5’ – (A/T)TT(G/C) – 3
protein involved in migration and resolution of Holliday junctions: RecG
-Can move Holliday junctions but not resolve
-Different from RuvAB -> can bind to 3-stranded junctions
-May be involved in backing up stalled replication forks at DNA damage
- Pri proteins could then function to reload DnaB to restart replication
How are linear DNA fragments recombined into the E. coli genome?
Recombination in E. coli different from higher organisms -> between small linear pieces of DNA and chromosome instead of 2 large DNAs; enzymes and mechanism very similar
Model for how linear DNA fragments are recombined into the E. coli genome using homol. recomb. and replication.
-RecBCD processes end of linear DNA forming 3’ ssDNA with Chi site, coated with RecA
-RecA mediates strand invasion and synapse formation
-Replication is initiated from invading DNA fragment
-Replication continues to terminus on one side or until met by replication fork from oriC at the other side
-After segregation, each daughter cell has different allele(s) if genetic differences exist between the recombined DNA and the chromosome
Many phages encode recombination functions necessary for ______ that mirror those of E. coli
-replication of their genomes
