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Lecture 6 Site specific recombination (Final).pptx
- 2. Contents Site-specific recombination Difference with Homologous Recombination DNA Recombinases Site-Specific Recombinases Classification Applications Biological roles of site specific recombination Summary References
- 3. Site-specific recombination Site-specific recombination, also known as conservative site-specific recombination, is a type of genetic recombination in which DNA strand exchange takes place between segments possessing at least a certain degree of sequence homology. In some cases the presence of a recombinase enzyme and the recombination site is sufficient for the reaction to proceed; In other systems a number of accessory proteins and/or accessory sites are required.
- 4. Difference with Homologous Recombination Homologous recombination occurs between DNA with extensive sequence homology anywhere within the homology. Site-specific recombination occurs between DNA with no extensive homology (although very short regions may be critical) only at special sites. The protein machinery for the two types of recombination differs too. Strand exchange during site-specific recombination occurs by precise break/join events and does not involve any DNA loss or DNA resynthesis.
- 5. DNA Recombinases DNA recombinases are widely used in multicellular organisms to manipulate the structure of genomes, and to control gene expression. These enzymes, derived from bacteria (bacteriophages) and fungi, catalyze directionally sensitive DNA exchange reactions between short (30– 40 nucleotides) target site sequences that are specific to each recombinase. Types include: Cre recombinase Hin recombinase Tre recombinase FLP recombinase
- 6. Site-Specific Recombinases Site-specific recombinases (SSRs) perform rearrangements of DNA segments by recognizing and binding to short DNA sequences (recombination sites), at which they cleave the DNA backbone, exchange the two DNA helices involved and rejoin the DNA strands. They are employed in a variety of cellular processes, including bacterial genome replication, differentiation and pathogenesis, and movement of mobile genetic elements.
- 9. Classification Based on amino acid sequence homology and mechanistic relatedness most site-specific recombinase are grouped into one of two families: 1. Tyrosine recombinase family 2. Serine recombinase family Member of the tyrosine recombinases include lambda- integrase, using attP/B recognition sites. Members of the serine recombinase family were known as resolvase / DNA invertases.
- 10. Tyrosine Recombinase Family Mechanism During strand exchange, the DNA cut at fixed points within the crossover region of the site releases a deoxyribose hydroxyl group. Recombinase protein forms a transient covalent bond to a DNA backbone phosphate. This phosphodiester bond between the hydroxyl group of the nucleophilic serine or tyrosine residue conserves the energy that was expended in cleaving the DNA. Energy stored in this bond is subsequently used for the rejoining of the DNA to the corresponding deoxyribose hydroxyl group on the other site.
- 11. Cont. Tyrosine recombinases, such as Cre or Flp, cleave one DNA strand at a time at points that are staggered by 6-8bp, linking the 3’ end of the strand to the hydroxyl group of the tyrosine nucleophile. Strand exchange then proceeds via a crossed strand intermediate analogous to the Holliday junction in which only one pair of strands has been exchanged.
- 14. The λ Integrase Site-specific Recombination Pathway λ Integrase is generally regarded as the founding member of the tyrosine recombinase family The site-specific recombinase encoded by bacteriophage λ (Int) is responsible for integrating and excising the viral chromosome into and out of the chromosome of its Escherichia coli host. Int carries out a reaction that is highly directional, tightly regulated, and depends upon an ensemble of accessory DNA binding proteins acting on 240 bp of DNA encoding 16 protein binding sites. This additional complexity enables two pathways, integrative and excisive recombination, whose opposite, and effectively irreversible, directions are dictated by different physiological and environmental
- 16. Serine Recombinase Family Serine recombinases is much less well understood. Classical members are gamma-delta and Tn3 resolvase, but also new additions like φC31-, Bxb1-, and R4 integrases, cut all four DNA strands simultaneously at points that are staggered by 2bp During cleavage, a protein-DNA bond is formed via a transesterification reaction in which a phosphodiester bond is replaced by a phosphoserine bond between a 5’ phosphate at the cleavage site and the hydroxyl group of the conserved serine residue
- 19. Applications Tracking cell lineage during development. Work was done in Drosophila using the Flp-FRT system Ablating a gene function during development. Inducing the expression of a gene at a specific time in development. Site-specific recombination has biotechnological applications Targeted mutation in a reverse genetic approach.
- 20. Biological roles of site specific recombination Phage use CSSR for integration of their genome to host. Use to alter gene expression by inversion CSSR maintaion structural intergity of circular dna molecules cylce of DNA replication,HR,and cell division Recombinases convert the multimeric circular DNA into monomers
- 21. Summary The genomes of nearly all organisms contain mobile genetic elements that can move from one position in the genome to another by either a transpositional or a conservative site-specific recombination process. Based on the mechanism followed, site-specific recombinase may belong to Tyrosine or Serine recombinase family. Many of the new arrangements of DNA sequences that site-specific recombination events produce have created the genetic variation crucial for evolution.
- 22. References Pierce BA. 2017. Genetics: A conceptual approach. W. H. Freeman & Cpmpany, USA, ISBN-10: 1319050964 Weaver, R. F., 2011 Molecular Biology 5th ed., ISBN: 9780073525327 https://www.ncbi.nlm.nih.gov/books/NBK26845/ https://en.wikipedia.org/wiki/Site-specific_recombination https://www.slideserve.com/vince/site-specific- recombination-transposition-of-dna https://www.researchgate.net/publication/26258501_Challe nging_a_Paradigm_the_Role_of_DNA_Homology_in_Tyro sine_Recombinase_Reactions/figures https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5710010/ https://www.brainscape.com/flashcards/lecture-6-site- specific-recombination-1703097/packs/3149010
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Hinweis der Redaktion
- Enzymes known as site-specific recombinases (SSRs) perform rearrangements of DNA segments by recognizing and binding to short, specific DNA sequences (sites), at which they cleave the DNA backbone, exchange the two DNA helices involved, and rejoin the DNA strands.
- In general recombination, DNA rearrangements occur between DNA segments that are very similar in sequence. Although these rearrangements can result in the exchange of alleles between chromosomes, the order of the genes on the interacting chromosomes typically remains the same. Whereas site-specific recombination, can alter gene order and also add new information to the genome. Site-specific recombination moves specialized nucleotide sequences, called mobile genetic elements, between nonhomologous sites within a genome. The movement can occur between two different positions in a single chromosome, as well as between two different chromosomes.
- Cre recombinase is a tyrosine recombinase enzyme derived from the P1 bacteriophage. The enzyme uses a topoisomerase I like mechanism to carry out site specific recombination events. The enzyme (38kDa) is a member of the integrase family of site specific recombinase and it is known to catalyse the site specific recombination event between two DNA recognition sites (LoxP sites). This 34 base pair (bp) loxP recognition site consists of two 13 bp palindromic sequences which flank an 8bp spacer region. The products of Cre-mediated recombination at loxP sites are dependent upon the location and relative orientation of the loxP sites.The enzyme requires no additional cofactors (such as ATP) or accessory proteins for its function. Hin recombinase is a 21kD protein composed of 198 amino acids that is found in the bacteria Salmonella. Hin belongs to the serine recombinase family (B2) of DNA invertases in which it relies on the active site serine to initiate DNA cleavage and recombination. Hin functions to invert a 900 base pair (bp) DNA segment within the salmonella genome that contains a promoter for downstream flagellar genes. Tre recombinase is an experimental enzyme that in lab tests has removed DNA inserted by HIV from infected cells. Through selective mutation, Cre recombinase which recognizes loxP sites are modified to identify HIV long terminal repeats (loxLTR) instead. As a result, instead of performing Cre-Lox recombination, the new enzyme performs recombination at HIV provirus sites. Flp-FRT recombination is a site-directed recombination technology, increasingly used to manipulate an organism's DNA under controlled conditions in vivo. It is analogous to Cre-lox recombination but involves the recombination of sequences between short flippase recognition target (FRT) sites by the recombinase flippase (Flp) derived from the 2 µ plasmid of baker's yeast Saccharomyces cerevisiae.
- Recombination sites are typically between 30 and 200 nucleotides in length and consist of two motifs with a partial inverted-repeat symmetry, to which the recombinase binds, and which flank a central crossover sequence at which the recombination takes place. The pairs of sites between which the recombination occurs are usually identical, but there are exceptions.
- A classical feature of the tyrosine recombinase family of proteins catalyzing site-specific recombination, as exemplified by the phage lambda integrase and the Cre and Flp recombinases, is the ability to recombine substrates sharing very limited DNA sequence identity. Decades of research have established the importance of this short stretch of identity within the core regions of the substrates. Since then, several new enzymes that challenge this paradigm have been discovered and require the role of sequence identity in site-specific recombination to be reconsidered. The integrases of the conjugative transposons such as Tn916, Tn1545, and CTnDOT recombine substrates with heterologous core sequences. The integrase of the mobilizable transposon NBU1 performs recombination more efficiently with certain core mismatches. The integration of CTX phage and capture of gene cassettes by integrons also occur by altered mechanisms. In these systems, recombination occurs between mismatched sequences by a single strand exchange.
- Recombination by tyrosine recombinases. Four recombinase monomers (blue ovals) bind to the substrates; two monomers are active (indicated by the tyrosine in position), and two are inactive. The active monomers cleave the first pair of DNA strands to form a 3 ‘ -phosphotyrosyl intermediate and free 5 ‘ -OH groups. Strand exchange results in an Holiday Junction intermediate. There is a conformational change, and the second pair of monomers becomes active, and they carry out the second set of DNA cleavages; the second round of strand exchanges and ligations results in the recombinant.
- Int recombinase is a heterobivalent DNA binding protein and each of the four Int protomers, within a multiprotein 400 kDa recombinogenic complex, is thought to bind and, with the aid of DNA bending proteins, bridge one arm- and one core-type DNA site.
- Bacteriophage lambda integrates into the chromosome of its Escherichia coli host by means of a site-specific recombination between a locus on the phage chromosome (phage att site) and a locus on the bacterial chromosome (bacterial att site). Integration requires a recombinase called lambda integrase and the integration host factor (IHF) encoded by a gene in the E. coli genome.
- Contrary to Tyr-recombinases, the four participating DNA strands are cut in synchrony at points staggered by only 2 bp (leaving little room for proofreading). Subunit-rotation (180°) permits the exchange of strands while covalently linked to the protein partner. The intermediate exposure of double-strand breaks bears risks of triggering illegitimate recombination and thereby secondary reactions. Contrary to members of the Tyr-class the recombination pathway converts two different substrate sites (attP and attB) to site-hybrids (attL and attR). This explains the irreversible nature of this particular recombination pathway, which can only be overcome by auxiliary "recombination directionality factors" (RDFs).