Module 5: Bacterial Genetics (Gene Transfer) Flashcards
Horizontal Gene Transfer
The movement of DNA between microbes
AKA. Lateral Gene Transfer
What is vertical gene transfer?
The INHERITANCE of genes from a direct ancestor
What are the main mechanisms of horizontal gene transfer?
1) Transformation
2) Conjugation
3) Transduction
4) Transposition
No matter the method of horizontal gene transfer, what is a necessary process for the transfer to occur successfully?
Some form of RECOMBINATION occurs to incorporate foreign DNA into the bacterial genome
DNA that enters a cell will be degraded and not maintained if…
it does not have a way of replicating
(not linked to usable origin of replication in host cell)
Chromosomes + plasmids vs DNA fragments in cells
–> What is the difference between what happens to them, why?
Chromosomes + Plasmids = Have origin/s of replication –> Replicate on their own; are maintained within cells
DNA Fragments = NO origin of replication –> Gets degraded and is NOT maintained within cell
What must DNA fragments “do” to maintain within a cell?
MUST incorporate into host genome to get replicated and therefore maintain presence in cell
Incorporation of DNA fragments into genome occurs via
Recombination
Recombination is a “spin off” of _____________.
It requires breaking and __________ DNA strands.
Recombination is a “spin off” of DNA repair mechanisms.
It requires breaking and rejoining DNA strands.
What are the main forms of recombination?
Homologous and NON-homologous
Homologous Recombination (define)
Process of 2 DNA segments with IDENTICAL or very SIMILAR DNA sequences pair up and exchange or replace some portion of their DNA
(Process of DNA exchange/replacement between 2 DNA segments that have some level of sequence homology)
Homologous Recomb. AKA =
CROSSOVER
RecBCD
An enzyme complex (RecB+ C + D) that functions in homologous recombination to:
1) UNWIND DNA
2) CLEAVE DNA
What type of enzyme complex is RecBCD?
Helicase-Nuclease
Helicase = unwinds, Nuclease = clips
What is responsible for initiating homologous recombination?
RecBCD activity on DNA
(forming the single stranded knick in the DNA)
RecA
Protein (ssDNA BP) that catalyzes DNA strand exchange (and invasion) in homologous recombination
What catalyzes holiday junction formation?
RecA
Catalyzes strand invasion + exchange leading to holiday junc. forming
Homologous Recombination: Process
1) RecBCD binds to foreign DNA fragment and begins to unwind the DNA
2) RecBCD encounters chi site while unwinding
3) Upon recognition of the chi site, RecBCD cuts ONE of the strands of the DNA at this site, producing a segment of SINGLE stranded DNA (on one side of the cut; the other side remains base paired)
4) RecA is recruited to the single stranded segment of the DNA and binds to it
5) RecA bound to the single strand scans chromosomal DNA for a homologous sequence (to the single stranded segment)
6) Once a homologous sequence is found, RecA invades the homologous region of the chromosome with the single strand it is bound to, annealing to the complementary chromosomal strand
–> Effectively, displaces a strand of the chromosomal DNA, making a single stranded segment of chromosomal DNA
7) Displaced chromosomal strand basepairs to the free bases on the foreign DNA fragment
8) Holiday Junction forms (crossed structure)
9) Holiday structure is cleaved in 2 spots (one cut made in each non-recombinant strand; “outer” strands that have not been cut in this entire process)
10) The free strands created from holiday junction cleavage are ligated to form 2 recombinant dsDNA molecules
What is invasion in terms of RecA function?
Strand bound to RecA is brought to a homologous segment of chromosomal DNA and aligned with the complementary strand in the homologous segment
–> The strand then base pairs to this complementary strand, kicking out the chromosomal strand it was already base paired with
Holliday Junction
4-way structure of DNA formed by exchange of strands between homologous DNA duplexes (dsDNA)
Where is the holliday junction cleaved?
Cleaved at ONE spot on BOTH non-recombinant strands (the strands that have remained in their original form thus far)
–> Cut in TWO places
What determines the TYPES of molecules that are produced from homologous recombination?
2 factors:
1) The TYPE (linear or circular) of DNA starting molecules
2) The NUMBER of crossovers that occur (1 or 2 free strands invading?)
What is the result of homologous recombination for:
Single cross-over between 2 circular molecules
= ONE circular recombinant molecule (that contains all the DNA of BOTH starting molecules)
–> No exchange; it’s like a summation
What is the result of homologous recombination for:
Double cross-over between 2 circular molecules
= TWO recombinant circular molecules (that have exchanged a segment of DNA)
–> Reciprocal exchange!