HOMOLOGOUS AND SITE-SPECIFIC RECOMBINATION

(1)

HOMOLOGOUS AND SITE-SPECIFIC RECOMBINATION

Dr. Nüket Yürür Kutlay

(2)

HOMOLOGOUS RECOMBINATION Homologous (general) recombination; genetic

exchange takes place between a pair of homologous DNA sequence ( similar or identical nucleotide

sequence)

The fundamental processes that catalyze homologous recombination are common to all cells (bacteria,

viruses, yeasts)

The total number of nucleotides is not changed.

(3)

Homologous recombination is used;

• to repair DNA (the most widespread use is in accurately repairing double-strand breaks)

• to exchange bits of genetic information between two different chromosomes to create new

combination

– Gene conversion

– Crossing-over in meiosis

• to assure an accurate chromosomal segregation.

(by playing an important mechanical role in

meiosis)

(4)

Figure 5-53 Molecular Biology of the Cell (© Garland Science 2008)

Repair of a single strand breakage

During the replication,

ehead of a replication fork

(5)

The initial strand invasion requires a free 3’ end

generated by nuclease that degrades the 5’ end of the complementary strand

One broken and one intact

daugher chromosome

(6)

• It takes place only between DNA duplexes that have extensive regions of sequence

similarity ( homology )

• Base-pairing is a requirement for a perfect

match and successful hom. rec.

(7)

Base-pairing interaction can be mimecked in a test tube by allowing a DNA double helix re-formed;

Random collision complementary base pairs have formed rapid

zippering formation of complete double helix

(8)

• Proteins have an important role in the base-pairing (to make it easier)

• Renaturation / hybridization

• Base pairing can not be done between two intact DNA double helices

– strand invasion

– RecA is bound to a DNA single strand

(9)

• For an unfolded conformation of annealing strands, SSB proteins are used (tightly and

cooperatively bind to sugar-phosphate backbone)

– DNA replication

– Homologous recombination

• Heteroduplex is an essential step in any

hom.rec.process. “a region of DNA helix formed from strands that originate from two different

DNA molecules.

(10)

Rad51(human homolog of bacterial RecA )helical filament

(11)

RecA filament can hold a single strand and double helix together,

and catalyze a multistep DNA synapsis.

(12)

in addition to recA(Rad51);

- Rad52 displaces SSB

protein, allowing the binding of Rad51

-

(13)

By specialized DNA helicases Unidirectional

After heterodublex region is formed, it is often enlarged by a process called branch migration

(14)

Repair of a double-strand breakage

Shortly after the DNA has been replicated and two sister chromatids are still held together.

Daughter DNA duplex can serve as the template for repair of the other

Without any loss or alteration of nucleotides at site of repair.

(15)

During hom. rec. a special DNA intermediate forms;Holliday junction(cross-strand exchange).

It contains four DNA strands shared between two DNA helices

Transiently present

(16)

Ruv A(green)tetramer

Ruv B(g)Hexamer helikaz (uses energy of ATP hydrolysis)

This junction must be cut for seperation; a process refered to as resolution.

Ruv C endonuclease RuvC (SLX1/SLX4, GEN1) or RecQ (BLM and TOPIIA)

(17)

As a result of meiotic recombination , hybrid chromosomes are

produced.

(18)

Budding yeast

Meiotic recombination begins with a programmed double strand break

Protruting 3’single strand ends

(19)

• Meiosis/DS break repair

– As well as proteins that function in DS break repair, several specific proteins serve in meiosis – In DS break repair, hom rec occurs between

identical DNA duplexes

In meiosis, hom rec occurs between Pat-Mat

chromosomal homologs

(20)

- Few of Spo11mediated DS breaks become crossover. The majority are resolved as non- crossover(%90)

- This choice is made before the Holliday junction are formed

- Crossover control; not closely placed. One

crossover on each arm of chromosome occurs

during meiosis

(21)

Event can be resolved as a crossover or a non-crossover, in both ways, heteroduplex regions remain

1000bp

Small percentage of mismatched base pairs can be tolerated.

Heteroduplex regions are potential gene conversion sites

(22)

Gene conversion:

- Corrected by mismatch repair machinery

- Strand which will removed is randomly selected

(23)

Homologous recombination is carefully regulated

• Loss of heterozygosity

• Recombination-based repair is prevented in the absence of DNA damage;

– BRCA1, BRCA2RAD51(binding and preventing its polymerization on DNA, thereby maintaining it in an inactive form /DNA damage; bring it and

release it)

• Too much or too little homologous recombination can

lead to cancer

(24)

Transposition(al rec)

Conservative site-specific recombination

• Mobile genetic elements

• Range in size; 100-10000bp

• Jumping genes/ selfish DNA/ mol.

parasites

• Had a profound effect on the shaping of modern genome

• A few of them in our DNA are still active

• Carries a unique set of genes

• Can alter gene order along chromosome

• Cause unusual types of

mutations

(25)

• Mobile elements that move by transposition are called transposons or transposable

elements

• They are only modestly selective in choosing their target side

• Move rarely

(26)

Three Major Classes of Transposoable elements

• DNA only transposons

• Retroviral like-retrotransposons

• Nonretroviral retrotransposons

(27)

DNA transposons

Once every 10

⁵

cell divisions

Horizontal gene transfer

(28)

DNA only transposons;

mode of movement

* Cut and paste pathway like VDJ

* Replicative pathway

(29)

• Life cycle of retrovirus (AIDS/HIV), integrase

• Retroviral-like retrotransposons

• Unlike viruses, they have no intrinsic ability

to leave their resident cell

(30)

Nonretroviral

retrotransposons

• mode of movement;

– Moves via an RNA

• Reverse transcriptase

and endonuclease

(31)

• 35% of genome

• L1(LINE):long interspersed nuclear element

• SINE: Short interspersed nuclear

element(Alu)

(32)

Together the LINEs and SINEs make up abouth 40% of human genome

Human genome contains all three types of transposon

(33)

Transposons and their activation situation varies according to species.

• In bacteria; mostly DNA transposons

• In yeast; mostly retro viral like retrotransposons

• In drozofila; all three transposons

(34)

Conservatif site-spesific recombination;

requires specialized DNA sequence

recombinases form transient high-energy covalent bonds

with DNA and use this energy to complete the DNA rearrangements

A bakteriofaj /B(Salmonella flagelin)

(35)

Molecular Biology of the Cell

Fifth Edition

Molecular Biology of the Cell

Fifth Edition

Chapter 5 DNA Replication, Repair, and Recombination

Homolog Recombination

Transposition and Concervative site-specific Recombination

Chapter 5 DNA Replication, Repair, and Recombination

Homolog Recombination

Transposition and Concervative site-specific Recombination

Alberts • Johnson • Lewis • Raff • Roberts • Walter Alberts • Johnson • Lewis • Raff • Roberts • Walter