DNA Recombination! Why all of us are

unique?

BINCY MARIAM YESUDASM 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

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

Mitotic and Meiotic Recombination

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

Gene shuffling allows favourable and non favourable alleles to be separated and tested in new assortments causing escape and spreading of favourable allele and elimination of unfavourable alleles- role in genetic diversity- natural selection and evolution

2. Mitotic recombination has roles in a) post replicational repair (repair of lesions at replication

forks and for restarting replication that stalled at these lesions)

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

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

Biological Roles for Recombination

1. Used to map genes on chromosomes- recombination frequency proportional to distance between genes

2. Making transgenic cells and organisms

Practical Uses of Recombination

DNA RECOMBINATIONTYPES OF 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.

HOMOLOGOUS RECOMBINATION

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(RecD and Rec B ) and a DNA-dependent ATPase activity

Rec BCD

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

MAIN FUNCTIONS

RecA 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.

PATHWAY OF HOMOLOGOUS RECOMBINATION

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 or homologous

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

Ends of the strands produced these cuts are displaced and pair with their complements on opposite duplex.

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

The exchange creates a cross bridged structure

RecBCD Pathway of Homologous Recomb. Part I: Nicking and Exchanging

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.

Part II: Branch Migration and Resolution

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.

RESOLUTION OF HOLLIDAY JUNCTION

EM of a Holliday Junction w/a few melted base pairs around junction

The two DNA molecules share limited homology 14-55 bp homology enzymes involved

E.g recombinasesE.g Integration of Lamda phage DNA into bacterial

genome 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

Site-specific recombination is guided by recombination enzymes that recognize short, specific nucleotide sequences present on one or both of the recombining DNA molecules.

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

Site-specific recombination

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 acceptorGene

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

NON-HOMOLOGOUS RECOMBINATION

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