BIOC 503 - DNA Recombination

DNA Recombination types

• homologous genetic recombination aka general recombination

• Site-specific recombination

• DNA transposition

General recombination

Exchange of sections of homologous, non-identical DNA molecules

1- homologous sequences are exchanged

2- process occurs wherever homologous sequences exist

3- resulting joints are staggered

4- no nucleotides are ultimately gained or lost at the exchange site, yet some mismatch is tolerated

meiosis recombination

• type of general recombination

• eukaryotic process

• prophase I

• chromosomes coding for the same gene products thus having similar DNA sequences (homologous) form a complex so DNA sequences touch (chiasma), forming a crossover point, and a small part of them is exchanged between the two.

1- double stranded break in one of the 2 homologs converted to double strand gap by exonucleases. Strands with 3’ ends degraded less compared to 5’ ends, leading to 3’ extensions

2- exposed 3’ end pairs with complement on intact homolog. Other strand of duplex displaced.

3- invading 3’ end extended by DNA polymerase and branch migration, generating Holiday intermediates (2 crossovers on generated DNA molecule)

4- further DNA replication replace DNA from missing sites on original double stranded break

5- cleavage of Holiday intermediates by nucleases generating either of the 2 possible recombination products

prokaryotic general recombination

• conjugation

• transducing phage

• transformation

conjugation

when a portion of the chromosome of a donor cell is transferred to a recipient cell, homologous sequences promote crossing over

transducing phage

when bacterial DNA is packaged into a phage particle and introduced into a different host cell via normal infection processes, and homologous sequences of host-bacterial DNA recombine.

transformation

when naked DNA from the environment is taken up by a bacterium, homologous sequences will promote recombination.

• source of antibiotic resistance

denaturation

provides single-stranded regions on one duplex that seek out like sequences on another duplex

renaturation

__ of complementary single-stranded regions of each homologue brings teh 2 interacting duplexes together in register

RecBCD

bacterial protein complex that has a helicase and exonuclease activity

• studied for associated recombination mechanism

• everything in bacteria starts with a double stranded break of DNA

1- Double stranded break of bacterial DNA

2- RecBCD binds to double stranded break, and acts as helicase to separate strands, and acts as exonuclease cleaving DNA until it reaches the CHI sequence.

3- CHI sequence stops 3’ to 5’ exonuclease activity, meaning that the 3’ to 5’ strands is not cleaved anymore, forming a tail with the chi sequence at the extremity

4- RecA filament recruited around 3’ to 5’ DNA tail in primary binding site, and binds double stranded DNA in the secondary binding site scanning along its sequence in search of homology

5- RecA promotes strands invasion once homology found, can involve 2 Holiday junctions. RecA then dissociates, DNA polymerase & DNA ligase fills in gap

6- RuvA (recognize Holiday junctions) and RuvB (2 per junction, ATPase) catalyze branch migration aka exchange of base pair

7- RuvC catalyzes Holiday junction resolution

RecA

another bacterial protein complex 

1- binds ssDNA cooperatively and directionally

2- ___-ssDNA complex binds to dsDNA using ATP as a cofactor

3- enhances process of branch migration using ATP hydrolysis for energy

non-crossover products

patch recombination products

• both recombination products cleaved in the same way

crossover products

splice recombination products

• junctions cleaved at different sites (one on DNA strand that did not cross over, one on the DNA strand that crossed over)

• DNA on either side of the region undergoing repair is recombined

Holiday Model

1- symmetric 1 strand nicks

2- swap strands forming heteroduplex and holiday junction

3- horizontal cut = non-crossover products

3- vertical cut = crossover products (all strands cut vertically, followed by rotation to untangle Holiday junction)

Holiday junction

structure forming when 2 cleavages and 2 ligations covalently lock the interacting duplexes to each other.

branch migration

process that elongates the region of interaction between the 2 duplexes

• breaking of old complementary pairs & making of new complementary pairs

BRCA-1/2

proteins that lead to breast or epithelial cancer when mutated as they are necessary to initiate double stranded break repair

site specific recombination

recombination process occurring ONLY at specific sequences

• enzyme is recombinase (catalyzes integration of one DNA into another, ex: lambda or transposon integration)

integrase

lambda recombinase for site specific recombination

transposase

enzyme recombinase for site specific transposons recombination

transposable elements

DNA sequences that do not have a fixed position in a genome & can move from place to place within it

• In both prokaryotes and eukaryotes

• require little to no sequence homology

• themselves encode for transposase enzyme which carry out the moving process

duplicated

Transposons are ___ before being inserted into their target sequences, much like a copy & paste mechanism (rather than cut and paste)

effects of transposition

• process involves duplication of target site

• DNA synthesis may be involved

• element itself may be duplicated

• process may restructure the chromosome by causing deletions or insertions, leading to gene inactivation

• gene may be activated due to moving a promoter into juxtaposition

• some transposons carry and confer antibiotic resistance

45

transposable elements make up to __% of the human genome