Types of Recombinase
- 1. CONTENTS Introduction Homologous & Site-specific recombination Recombinases involved in homologous recombination • RecBCD • RecA Recombinases involved in Site-specific recombination Types of site-specific recombinases • Serine recombinases - Hin recombinase 1
- 2. • Tyrosine recombinases - Cre recombinase - Lambda recombinase - Xer recombinase Conclusion References 2
- 3. INTRODUCTION Recombinases are genetic recombination enzymes . An enzyme that catalyzes the exchange of short pieces of DNA between two long DNA strands , particularly the exchange of homologous regions between the paired maternal & paternal chromosomes . These enzymes derived from bacteria & fungi , catalyze directionally sensitive DNA exchange reactions between short (30 – 40 nucleotides) target site sequences that are specific to each recombinase . 3
- 4. The recombinases bring these specific site together to form a protein – DNA complex bridging the DNA sites known as the synatic complex . Within synaptic complex , the recombinase catalyzes the clevage & rejoining of the DNA molecules either to invert a DNA segment or to move a segment to a new site . 4
- 5. HOMOLOGOUS & SITE – SPECIFIC RECOMBINATION Homologous recombination is the process where by DNA segments that are identical or similar to each other break & rejoin to form a new recombination . It involves an exchange between DNA segments that are similar or identical in their DNA sequences . During site – specific recombination , non-homologous DNA segments are recombined at specific sites . 5
- 6. RCOMBINASES INVOLVED IN HOMOLOGOUS RECOMBINATION Homologous recombination is found in all species , & requires the participation of many proteins & the types of proteins that participate in the steps are very similar . This process is best understood in Escherichia coli . 6
- 7. RECBCD The term Rec indicates that these proteins are involved with recombination . RecBCD is a protein complex composed of the RecB , RecC & RecD proteins . The RecBCD complex plays an important role in the initiation of recombination involving double-strand breaks . In this process , RecBCD recognizes a double-strand break within DNA & catalyzes DNAunwinding & strand degradation . 7
- 8. The action of RecBCD produces single-stranded DNA ends that can participate in strand invasion & exchange . The single-stranded DNA ends are coated with single-stranded binding protein to prevent their further degradation . The RecBCD complex can also create breaks in the DNA at sites known as chi-sequences . In E.coli , the chi sequence is 5’-GCTGGTGG-3’ . 8
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- 10. RECA The function of the RecA protein is to promote strand invasion . To accomplish this task , it bins to the single-stranded ends of DNA molecules generated from the activity of RecBCD . A large number of RecA proteins bind to single- stranded DNA , forming a structure called a filament . During strand invasion , this filament makes contact with the unbroken chromosome . Initially , this contact is most likely to occur at non- homologous regions . 10
- 11. The contact point slides along the DNA until it reaches a homologous regions . Once a homologous site is located , RecA catalyzes to displacement of one DNA strand & the invading single-stranded DNA quickly forms a double helix with the other strand This results in a D-loop . RecA proteins mediate the movement of invading strand & the displacement of the complementory strand . This occurs in such a way that the displaced strand invades the vacant region of the broken chromosome . 11
- 12. RCOMBINASES INVOLVED IN SITE-SPECIFIC RECOMBINATION Conservative site-specific recombination (CSSR) is a recombination between two defined sequence elements . Site-specific recombinases cleave& rejoin DNA using a covalent Protein-DNA intermediate . It is this mechanistic feature that contributes the “conservative” to the CSSR name :- it is called “conservative” because every DNA bond is resealed by the recombinase . 12
- 13. TYPES OF SITE-SPECIFIC RECOMBINASES There are two families of conservative site-specific recombinase :- the serine recombinases & the tyrosine recombinases . For the serine recombinase , the side chain of a serine residue within the protein’s active site attacks a specific phosphodiester bond in the recombination site . This reaction introduces a single stranded break in the DNA & simultaneously generates a covalent linkage between the serine & a phosphate at this DNA cleavage site . Likewise , for the tyrosine recombinases , it is the side chain of the active-site tyrosine that attacks & then becomes joined to the DNA . 13
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- 15. SERINE RECOMBINASES Serine recombinases introduce Double-strand breaks in DNA & then swap strands to promote recombination . The serine recombinases cleave all four strands prior to strand exchange . One molecule of the recombinase protein promotes each of these cleavage reactions , therefore a minimum of four subunits (i-e a tetramer) of the recombinase is required . 15
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- 17. HIN RECOMBINASE Hin belongs to the serine recombinase family of DNA invertase & is a 21KD protein composed of 198 amino acids that is found in the bacteria Salmonella . The Salmonella Hin recombinase inverts a segment of the bacterial chromosome to allow expression of two alternative sets of genes . The chromosomal region inverted by Hin is about 1000 bp & is flanked by specific recombination sites called hix L (on the left) & hix R (on the right) . 17
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- 19. TYROSINE RECOMBINASES Tyrosine recombinases break & rejoin one pair of DNA strand at a time . In contrast to the serine recombinases , the tyrosine recombinases cleave & rejoin two strands first & only then cleave & rejoin the other two strands . 19
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- 21. CRE RECOMBINASE Cre is an enzyme encoded by phage P1 , which function to circularize the linear phage genome during infe-ction . The recombination sites on the DNA where Cre acts , are called lox sites . Cre-lox is a simple example of recombination by the tyrosine recombinase family , only Cre protein & the lox sites are needed to complete recombination . 21
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- 23. LAMBDA RECOMBINASE When bacteriophage lambda infects a host bacterium , a series of regulatory events result either in establishment of the quiescent lysogenic state or in phage multiplication , a process called lytic growth . Establishment of a lysogen requires the integration of the phage DNA into the host chromosome . Likewise , when the phage leaves the lysogenic state to replicate & make new phage particles , it must excise its DNA from the host chromosome . The analysis of this integration /exision reaction provided the first molecular insights into site-specific recombination . 23
- 24. To integrate the lambda integrase protein catalyzes recombination between two specific sites , known as the att or attachment sites . The att P site is on the phage DNA (P for phage) & the att B site is in the bacterial recombinase & the mechanism of strand exchange follows the pathway described for the Cre protein . Unlike Cre recombination , lambda integration requires accessory proteins to help the required Protein-DNA complex to assemble . 24
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- 26. XER RECOMBINASE Xer is a member of the tyrosine recombinase family . The Xer recombinase catalyzes the monomerization of bacterial chromosomes & of many bacterial plasmids . Xer is a heterotetramer containing two subunits of a protein called Xer C & two subunits of aprotein called Xer D . Both Xer C & Xer D are tyrosine recombinases but they recognizes different DNA sequences . 26
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- 28. CONCLUSION Recombinases are widely used in organisms to manipulate the structure of genomes & to control gene expression . Different recombinases involved in recombination processes are RecA , Cre ,Hin , Xer , Lambda recombinases . 28
- 29. REFERENCES Genetics Analysis & Priciples – Robert J.Brooker (4th Edition) Molecular Biology of Gene – Watson (5th Edition) www.sciencedirect.com/.../Recombinase www.proteopedia.org/.../Recombinase-A https://www.ncbi.nlm.nih.gov/.../173646... 29
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