DNA Recombination!

Why all of us are

unique?

BINCY MARIAM YESUDAS

M Sc BOTANY

Homologous recombination in E.coli

Site specific recombination

Gene conversion

Non-Homologous Recombination

Two DNA molecules exchange genetic information, resulting in the production of a new combination of alleles.

New allele/gene combinations are created by crossing over that occur during meiosis.

Mitotic recombination also generate new genes .

Plays an important role in DNA damage repair

DNA recombination studies used to map genes on chromosomes.

Recombination can occur both during mitosis and meiosis

Only meiotic recombination serves the important role of re-assorting genes

Mitotic recombination may be important for repair of mutations in one of a pair of sister chromatids

1. Generating new gene/allele combinations (crossing over during meiosis)

Gene shuffling allows favourable and non favourable allelesto be separated and tested in new assortments causingescape and spreading of favourable allele and elimination ofunfavourable alleles- role in genetic diversity- naturalselection and evolution

2. Mitotic recombination has roles in

a) post replicational repair (repair of lesions at replicationforks and for restarting replication that stalled at theselesions)

b) Generating new genes (e.g., Immuno- globulinrearrangement) also known as somatic recombination

c) Yeast mating type switching (sequence at an active locusreplaced by a sequence from a silent locus)

1. Used to map genes on chromosomes

- recombination frequency proportional to

distance between genes

2. Making transgenic cells and organisms

DNA RECOMBINATION

Generalized

Non-homologousHomologous

site specific

It is a physical phenomenon where exchange of sequence occur with no net gain or loss of nucleotides

It is based on sequence complementarity.

- Occurs between sequences that are nearly identical (e.g., during meiosis)

Homologous recombination is extensively studied in E.coli.

At least 25 proteins are involved in recombination in E.coli.

Recombination in E coli

Enzymes involved are

•Rec BCD

•Rec A

•Ruv A , B, C

•Ruv G

A Complex Enzyme complex with endonuclease

and helicase activity.

1. Endonuclease subunits (RecBC) that cut one DNA strand close to Chi sequence.

2. DNA helicase activity in presence of a SSB(RecDand Rec B ) and a DNA-dependent ATPaseactivity

Essential for 99% of recombination events occurring at double-stranded breaks in bacteria.

Binds double stranded break Unwinds and degrades DNA Pauses at chi sequence Loads RecA on 3’ ssDNA extensions

38 kDa protein

Catalyzes strand exchange, also an ATPase

Also binds DS DNA, but not as strongly as SS

Involved in SOS response

Catalyses in strand transfer

Eukaryotes have multiple homologs of RecA

Rad51 is best studied

RecA can generate Holliday junction

By its strand transfer &displacement reactions.

Chi site (Χ-site)

• Recombination hotspot

• Modifies RecBCD enzymatic activity

5’ GCTGGTGG 3’

• 1009 chi (Χ) sites in E. coli genome.

• Recombination start point 10 kb right to the

x-site

Most popular model to explain homologous recombination.

Holliday model

It was proposed by Robin Holliday.

Holliday Model

R. Holliday (1964)

- Holliday Junctions form during recombination

- HJs can be resolved 2 ways, only one produces true

recombinant molecules

It begins with two paired DNA duplexes orhomologous

In each of which an endonuclease introduces a singlestranded nick at an identical position chromosomes.

Ends of the strands produced these cuts aredisplaced and pair with their complements on oppositeduplex.

A ligase seals the loose ends creating hybrid duplexescalled heteroduplex DNA molecules.

The exchange creates a cross bridged structure

The position of this cross bridge can move down the chromosome by the branch migration.

Ruv B is a DNA helicase that catalyzes branch migration.

It occur as a result of a zipper like action as Hydroygen bonds are broken.

Then reformed b/w complementary bases of the displaced strands of each duplex.

Migration yields an increased length of heteroduplex DNA on both homologs.

The duplex will separate ,bottom portions rotate about180*.

Now the duplex form a planar structure called a X-form That is Holliday junction (Chi form)

Two strands on opposite homologs previously uninvolved in the exchange are now nicked by an endonuclease

Then ligation occurs

Recombinant duplexes are created.

RecBCD Pathway

of Homologous

Recombination

Part II: Branch

Migration and

Resolution

Resolution of H.J is achieved by Ruv protein.

RuvA tetramer binds to HJ (each DNA helix between subunits), forces it into rotate about 180 to form square planar conformation

Resolution of H.J is catalysed by RuvC : resolvase

It is an endonuclease that binds to HJ as a dimer .

That cuts 2 strands of HJ.

It decide whether to cut horizontally or tranverse cut at the Holliday junction.

The two DNA molecules share limited homology

14-55 bp homology enzymes involved

E.g recombinases

E.g Integration of Lamda phage DNA into bacterialgenome

O ‘core region - 15 bp sequence that is common between phage DNA and bacterial chromosome

Site-specific recombination alters gene order, which would not happen during general recombination

Site-specific recombination is guided byrecombination enzymes that recognize short, specificnucleotide sequences present on one or both of therecombining DNA molecules.

The best example of the conservative site-specificrecombination is Bacteriophage lambda.

Bacterial viruses (bacteriophages) reproduce by a lytic or a lysogenic cycle

• Phage – temperate

• Bacteria – lysogenic

Life The Science of Biology, 7th Edition

Lysogenic cycle involves integration of phage into the host chromosome by SITE-

SPECIFIC RECOMBINATION

Molecular Biology of the Gene, 5th Edition

Gene Conversion

A special type of homologous recombination

Non-reciprocal transfer of genetic material from a ‘donor’

sequence to a highly homologous ‘acceptor’ sequence

Initiated by double strand DNA (dsDNA) breaks

5’ > 3’ exonucleases

Outcome: portion of ‘donor’ sequence copied to

‘acceptor’and original ‘donor’ copy unchanged

donor acceptor

Gene

Conversion

Gene Conversion is not uncommon

Yeast mating type switch (MAT) genes

Human repetitive sequence elements (Alu and LINE-1 sequences)*

Human gene families (e.g. MHC alleles, Rh blood group antigens,

olfactory receptor genes)

Chicken B cells Ig gene diversification

Pathogen clonal antigenic variation (e.g. African Trypanosomes

and Babesia bovis)

Here DNA elements moves from one site to the another .

Little sequence similarity is involved.

Transposition of genes takes place

Ability of genes to change position on chromosome.

A transposable element is removed from site & inserted into a second site in the DNA.

A transposable element (TE, transposon ) is a DNA sequence that can change its position within the genome.

sometimes creating or reversing mutations and altering the cell's genome size

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