Recombination dna repair bincy
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Transcript of Recombination dna repair bincy
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DNA Recombination! Why all of us are
unique?
BINCY MARIAM YESUDASM Sc BOTANY
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Homologous recombination in E.coli
Site specific recombination
Gene conversion
Non-Homologous Recombination
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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
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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
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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
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1. Used to map genes on chromosomes- recombination frequency proportional to distance between genes
2. Making transgenic cells and organisms
Practical Uses of Recombination
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DNA RECOMBINATIONTYPES OF RECOMBINATION
Generalized
Non-homologousHomologous
site specific
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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
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Recombination in E coli Enzymes involved are
•Rec BCD•Rec A•Ruv A , B, C •Ruv G
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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
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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
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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.
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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
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Most popular model to explain homologous recombination.
Holliday model
It was proposed by Robin Holliday.
PATHWAY OF HOMOLOGOUS RECOMBINATION
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Holliday Model
R. Holliday (1964)
- Holliday Junctions form during recombination
- HJs can be resolved 2 ways, only one produces true recombinant molecules
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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
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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
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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.
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RecBCD Pathway of Homologous Recombination
Part II: Branch Migration and Resolution
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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
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EM of a Holliday Junction w/a few melted base pairs around junction
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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
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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
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Bacterial viruses (bacteriophages) reproduce by a lytic or a lysogenic cycle
• Phage – temperate• Bacteria – lysogenic
Life The Science of Biology, 7th Edition
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Lysogenic cycle involves integration of phage into the host chromosome by SITE-SPECIFIC RECOMBINATION
Molecular Biology of the Gene, 5th Edition
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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
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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)
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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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