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
