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Home , Homologous recombination

Homologous Recombination • repair of dsDNA damage • recombination between homologous

STEP 1 create ssDNA with free 3’OH ssW1 dsC2-W2 STEP 2 find by strand exchange: ssW2 dsC2-W1 STEP 3 extend region of strand exchange beyond initial homology

STEP 4 resolve junction of dsDNAs to reestablish 2 separate chromosomes

DNA break repair by homologous recombination

This requires specialized factors: a protein helps the ssDNA region to find the homologous dsDNA in order to trade base-pairing. STEP 1: Create ssDNA with free 3’ OH

Eukaryotes typically load a 5’-3’ at a dsDNA break.

Also possible to nick DNA then load a :

5’ 3’

3’ 5’ 3’ 5’

3’ 5’

In E. coli, homologous recombination is induced by RecBCD

RecB and RecD are with opposite polarity. They load as a complex with each other and RecC at a break. Rec B is also a ; it cuts both single strands generated by the helicases UNTIL it encounters (running in the right polarity) the ‘chi’ site, at which point it leaves the strand with the free 3’ OH alone and continues to degrade the other strand. STEP 2: Strand exchange to find homology

E. coli uses RecA

The ssDNA in a RecA filament threads past dsDNA, with base flipping from the dsDNA acting to sample homology with the ssDNA. Binding to RecA induces underwinding of the DNA, which encourages bases to flip back and forth between the two possible partner strands WITHOUT additional input of energy.

Model for DNA strand exchange mediated by RecA. A three-strand reaction is shown. (a) RecA protein forms a filament on the single-stranded DNA. (b) A homologous duplex incorporates into this complex. (c) One of the strands in the duplex is transferred to the single strand originally bound in the filament. The other strand of the duplex is displaced.

Important features of RecA: • A monomer binds ~3 nt or bp • Cooperative filament assembly 5’-3’ • Prefers to form filament on ssDNA, but once formed, the filament will take up dsDNA at a second site • Filament has 18.6 bp DNA/turn: bp are de-stabilized and can rapidly exchange between two bound • Bound ATP increases RecA DNA affinity, ATP hydrolysis decreases affinity for DNA Human cells have the RecA-like protein Rad51; Rad51 needs extra help

STEP 2 Products

Each ssDNA strand exchanged generates a . Several series of steps are possible, so ONLY consider the model junction below.

3’ **Stable strand C1 W1 exchange by RecA 5’ 3’ 5’ 3’ requires >50 bp W2 C2 of PERFECT homology 3’ STEP 3: Extend region of strand exchange “” of the Holliday junction

3’ C1 W1 5’ 5’ W2 C2 3’

C1 W1 heteroduplex W2 C2

C1 W1 OR heteroduplex W2 C2

The Holliday junction is held in square-planar configuration by a sandwiching octamer of RuvA

C1 C1 W1 W2 W2-C1 heteroduplex parental heteroduplex W2 W1 C2 C2

parental W2 C1 C2 W1 parental

C2-W1 heteroduplex RuvA (in green) maintains Holliday heteroduplex junction geometry, recruits RuvB W2-C1 RuvB (in white) is a hexameric helicase; it extends the heteroduplex

parental parental W2 C1 C2 W1

**RuvB is ATP-powered: heteroduplex formation can proceed WITHOUT perfect homology, over C2-W1 long (>1 kb) regions heteroduplex

Holliday junction resolution: the RuvC (E. coli) It must nick BOTH Crick strands OR BOTH Watson strands to separate the two duplex DNAs (different chromosomes) W2-C1 heteroduplex

C1 C1 W1 W2

W2 W1 C2 C2

Cut at BOTH W2 C1 thin OR BOTH C2 W1 thick arrows parental parental

C2-W1 heteroduplex (eukaryotic equivalents of RuvABC have been purified but their identity is not certain) C1 W2 C1 C1 W1 W1

W2 W2 C2 C2 W1 C2

C1 W1 W2 C2 W1 C2 C1 W2 C1 W1 W2 C2 100% chance of some heteroduplex 50% chance of recombinant ends OR C1 (exchange of arms) W2 3’ C1 C1 W1 W1 5’ 5’ W2 W2 C2 C2 3’ W1 C2

Gene conversion can make heterozygous loci homozygous (called loss of heterozygousity or LOH) Products of HR (shown with recombinant ends, but note that central heteroduplex is present even with parental ends): Large-scale rearrangments by inappropriate HR

RuvC cleaves to give back parental ends

RuvC cleaves to give recombinant ends: deletion, inversion and translocation events

Homologous recombination with RECOMBINANT ENDS that occurs between duplicated genes (or other duplicated loci) can result in chromosome deletion, inversion and translocation events

DELETION

INVERSION

TRANSLOCATION

chromosome 2 Site-specific recombination

Protein-DNA recognition at sites with a specific sequence The two sites ‘synapse’ then all four strands are cut in series to exchange the original ends for recombinant ends.

Performed by a tetramer of a site-specific . The enzyme active site tyrosine forms a covalent protein-DNA intermediate like a topoisomerase, so the recombination reaction is reversible with no need for DNA ligase.

Site-specific recombination The only difference between the reactions in (A) and (B) is the relative orientation of the two DNA sites (indicated by arrows) at which a site-specific recombination event occurs. Why bother with site- specific recombination? A surface contour model of Cre recombinase bound to a recombination intermediate. The protein has been rendered transparent so that the bound DNA is visible.

Lambda phage hides in the E. coli chromosome by integration: attB (bacterium): 25 bp

attP (phage): 240 bp attL (left) attR (right)

Salmonella evades the immune system by changing gene expression:

gene on (gene off) Example of a DNA transposon

IS = “insertion sequence” for the mode of its discovery Non-replicative (cut-and-paste) transposition. DNA-based transposons have an inverted repeat sequence at their ends, and any DNA between them can be moved. Transposase multimers make a blunt double-stranded cut at the edge of the inverted repeat termini. Transposase also has a second binding site for DNA that is not sequence-specific, which it uses to bind an insertion target site and make a staggered double-stranded cut. Transposase bound to the transposon ends reverses its cleavage reaction to ligate the transposon DNA to the target site ends, but a gap remains on each side of the inserted DNA due to the staggered target site cut. Repair synthesis is required to rejoin the broken donor chromosome and to fill in the target site gaps.

Different transposase enzymes make different types of staggered cuts.

Depending on order of the next steps, transposition can result in transposon movement or transposon retention at the donor site and insertion elsewhere as well.

If transposase nicks the donor site ends rather than cutting both strands at once then donor 3’ ends join target 5’ ends, target 3’ ends prime replication and result in duplication of the transposon. The resulting donor-target fusion is fixed by the activity of a transposon-encoded site-specific recombinase or ‘resolvase’.

Importantly, even if the transposon departs from the donor site, the target site direct repeat is left behind (this is mutagenic). Antibiotic resistance genes were found in bacterial transposons, suggesting that ‘selfish’ mobile DNA elements can carry useful genes

LTR () retrotransposons transposase

a virally encoded integrase enzyme pastes the into the host chromosome (like a transposase second step).

The life cycle of an LTR (like HIV) A Non-LTR retrovirus lifecycle

(like the LINE elements that constitute 21% of our genome)

Additional occurs from homologous recombination between transposable elements DELETION

INVERSION

chromosome 1 TRANSLOCATION

chromosome 2 Some examples of single-gene diseases

Manipulating the genome using endogenous DNA repair to perform

A B C D E F G

a b c d e f g

A B C D E F G

a b c D e f g X ray-induced break !

Homologous Recombination

sister chromatid !

DNA replaced with same sequence

induced break !

Induce a dsDNA break at the mutation site to be repaired.

donor DNA ! Provide a template for homologous recombination

target replaced with donor DNA sequence !

(plasmid without origin is lost) Zinc Finger Protein (ZFP)-

Site-specific DNA breaks could be used for gene correction or gene disruption

DSB

+ donor DNA? no yes

non-homologous end-joining homologous recombination

deletion? gene conversion