• repair of dsDNA damage

• recombination between homologous chromosomes

STEP 1 create ssDNA with free 3’OH

STEP 2 find homology by strand exchange:

STEP 3 extend region of strand exchange

beyond initial homology

STEP 4 resolve junction of dsDNAs to

reestablish 2 separate chromosomes

ssW1 dsC2-W2

ssW2 dsC2-W1

Homologous Recombination

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

3’

5’

5’

3’

3’

5’

5’

3’

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

Also possible to nick DNA then load a helicase:

In E. coli, homologous recombination is induced by RecBCD

RecB and RecD are helicases with opposite polarity.

They load as a complex with each other and RecC at a break.

Rec B is also a nuclease; 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.

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.

STEP 2: Strand exchange to find homology

E. coli uses RecA

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 DNAs

• 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 Holliday junction.

Several series of steps are possible,

so ONLY consider

the model junction below.

C1

W1

W2

C2

3’5’

3’5’

3’

3’**Stable strand

exchange by RecA

requires >50 bp

of PERFECT homology

STEP 3: Extend region of strand exchange

“Branch Migration” of the Holliday junction

C1

W1

W2

C2

5’

5’

3’

3’

C1

W1

W2

C2

C1

W1

W2

C2

OR

heteroduplex

heteroduplex

The Holliday junction is held in square-planar

configuration by a sandwiching octamer of RuvA

C1

W1

W2

C2

W1

C2

C1

W2 W2-C1

C2-W1

W2

C2

C1

W1

heteroduplex

heteroduplex

parental

parental

parental heteroduplex

W2-C1

C2-W1

W2

C2C1

W1

heteroduplex

heteroduplex

parental parental

RuvA (in green) maintains Holliday

junction geometry, recruits RuvB

RuvB (in white) is a hexameric helicase;

it extends the heteroduplex

**RuvB is ATP-powered:

heteroduplex formation

can proceed WITHOUT

perfect homology, over

long (>1 kb) regions

Holliday junction resolution: the endonuclease RuvC (E. coli)It must nick BOTH Crick strands OR BOTH Watson strandsto separate the two duplex DNAs (different chromosomes)

C1

W1

W2

C2

W1

C2

C1

W2

W2-C1

C2-W1

W2

C2

C1

W1

heteroduplex

heteroduplex

parental parental

Cut at BOTH

thin OR BOTH

thick arrows

(eukaryotic equivalents of RuvABC have been purified but their identity is not certain)

C1

W1

W2

C2

C1

W1

W2

C2

C1

W2

W1

C2

W2

C2

C1

W1

C1

W2

C1

W1

W2

C2

5’

5’

3’

3’C1

W1

W2

C2

C1

W2

W1

C2

C1

W1

W2

C2W1

C2

OR

100% chance of some heteroduplex

50% chance of recombinant ends

(exchange of chromosome arms)

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):

RuvC cleaves to giveback parental ends

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

Large-scale genome rearrangments by inappropriate HR

Homologous recombination with RECOMBINANTENDS that occurs between duplicated genes

(or other duplicated loci) can result in chromosomedeletion, inversion and translocation events

chromosome 1

chromosome 2

DELETION

INVERSION

TRANSLOCATION

Protein-DNA recognition at sites with a specific sequenceThe 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 recombinase.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

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.

attB (bacterium): 25 bp

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

gene on (gene off)

Salmonella evades the immune system by changing gene expression:

Lambda phage hides in the E. coli chromosome by integration:

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.

Importantly, even if the transposon

departs from the donor site, the

target site direct repeat is left

behind (this is mutagenic).

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’.

Antibiotic resistance genes were found in bacterial transposons,

suggesting that ‘selfish’ mobile DNA elements can carry useful genes

LTR (long terminal repeat) retrotransposons

a virally encoded integrase

enzyme pastes the virus into

the host chromosome (like

a transposase second step).

transposase

The life cycle of

an LTR retrovirus

(like HIV)

A Non-LTR

retrovirus

lifecycle

(like the LINE elements

that constitute 21%

of our genome)

chromosome 1

chromosome 2

DELETION

INVERSION

TRANSLOCATION

Additional mutagenesis occurs from homologous

recombination between transposable elements

Some examples of single-gene diseases

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

Manipulating the genome using endogenous

DNA repair to perform gene conversion

X ray-induced break !

sister chromatid !

DNA replaced with same sequence

Homologous

Recombination

target replaced withdonor DNA sequence !

induced break !

donor DNA !

(plasmid without origin is lost)

Provide a plasmid template

for homologous recombination

Induce a dsDNA break at the

mutation site to be repaired.

Zinc Finger Protein (ZFP)-Nucleases

DSB

+ donor DNA? no yes

non-homologous end-joining homologous recombination

deletion? gene conversion

Site-specific DNA breaks could be used for

gene correction or gene disruption