Site-Specific Recombination OF MOLECULER GENETICS.pptx

MOLECULER GENETICS HANDMADE PPT

1. Site-Specific Recombination and Transposition of DNA MOLECULAR BIOLOGY OF THE GENE S E V E N T H E D I T I O N JAMES D. WATSON ALEXANDER GANN Institute of Technology University of California, Berkeley STEPHEN P. BELL RICHARD LOSICK Massachusetts Institute of Technology Harvard University

2. Replication, repair, and homologous recombination (High Fidelity)+ make sure that the genome remains identical in next generation. But Some genetic processes rearrange DNA seq & more dynamic genome structure. Genetic recombination—2 classes: ( Imp DNA rearrangements) Conservative site-specific recombination ( CSSR ) Transpositional recombination ( Transposition ) CSSR : Recombination b/w 2 defined seq elements (Fig. 12-1). Transposition : Recombination b/w specific seq & nonspecific DNA sites: Biological process introduces Transposition : Insertion of viral genomes to host DNA in infection (Inversion of DNA segments to alter gene structure, & movement of transposable elements). DNA rearrangements on chromosome structure cause function impact: Spontaneous mutation. A. Viral infection+ B. Vertebrate immune system development dep on these specialized DNA rearrangements.

3. Fig. 12-1 Two classes of genetic recombination. (Top panel) Example of site-specific recombination (CSSR) : R ecombination b/w red & blue recombination sites inverts DNA segment carrying the A and B genes. (Bottom panel) Transposition in which red transposable element excises from gray DNA, & inserts into an unrelated site in blue DNA.

4. Recombinases (Proteins) recognize specific seq for recombination within DNA molecules. Recombinases: Bring specific sites together: Protein–DNA complex bridging the DNA sites: Synaptic complex . Within synaptic complex : Recombinase catalyzes cleavage & rejoining of DNA molecules. CSSR+ Transpositions: Controlled: No drastic danger to the cells.

5. CONSERVATIVE SITE-SPECIFIC RECOMBINATION (CSSR) Recombination: Rearrangements of DNA Molecules. DNA segment that will be moved carries specific short seq: Recombination sites . Recombination sites: 20 bp (short seq)/so, although they may be much longer and carry additional seq. CSSR generate 3 diff DNA rearrangements ( Fig. 12-3 ): (1) insertion of DNA segment at specific site (as occurs during bacteriophage λ DNA integration), (2) deletion of DNA segment (3) Inversion of DNA segment. Whether recombination results in DNA insertion, deletion/inversion dep on Recombinase recognition sites organization on DNA molecule/Molecules participate in recombination. Rearrangement in CSSR always happens b/w 2 recombination sites: Whether the sites are present on same DNA/2 different DNA molecules: Impact will be on DNA: Present in b/w recombination sites . Lambda bacteriophage integrates its DNA to E. coli DNA by using this process (later). Recombination sites carry 2 classes of seq elements: seq specifically bound by recombinases & seq where DNA cleavage and rejoining occur.

6. Fig. 12-2 Integration of the λ genome into chromosome of the host cell. DNA exchange occurs specifically b/w recombination sites on 2 DNA molecules. The relative lengths of λ & cellular chromosomes not shown to scale.

7. Fig. 12-3 . 3 CSSR recombination . In each case, it is red DNA segment: moved/rearranged during recombination. A, B, C, D, X, and Y denote genes that lie within diff DNA segments. Darker red, & blue boxes represent recombinase-recognition seq, & black arrows show crossover regions. Together these seq elements form recombination sites.

8. To understand these rearrangements we must first ..find Seq elements in recombination sites (Fig. 12-4). 2 Imp regions in pair in these sites are (a) A pair of recombinase recognition seq. The crossover region, where DNA cleavage, & rejoining occur. *Recognition regions are symmetrically paired but crossover regions are asymmetric, so this region give a sense of direction /orientation to the recombination sites

9. Fig. 12-4 Structures involved in CSSR. The pair of symmetric recombinase recognition seq flanks crossover region where recombination occurs. S ubunits of recombinase bind these recognition sites. Note that the seq of crossover region is not palindromic, resulting in intrinstic asymmetry to recombination sites.

10. If the sites are on same DNA molecule then: either an inverted repeat/direct repeat manner. Recombination b/w pair of inverted sites will invert the DNA segment b/w 2 sites (Fig. 12-3). When sites are on 2 diff DNA molecules: Then DNA will be added on each other. Recombination b/w sites organized as direct repeats delete DNA segment b/w 2 sites (12-3).

11. There are 2 families of conservative site-specific recombinases: Ser & Tyr recombinases. M echanism: When they cleave DNA, a covalent protein– DNA intermediate is generated. In Ser/Tyr recombinase: Its Ser/Tyr residue of active site of enzyme : attach phosphodiester bond & make covalent linkage with phosphate. Covalent protein–DNA intermediate conserves energy. The same energy can be used in the rejoining reaction. For reversal, OH group from cleaved DNA attacks covalent bond: Links the protein to DNA. This process covalently seals DNA break, & regenerate free (non–DNA bound) recombinase (Fig. 12-5). CSSR as: Conservative because every DNA bond that is broken during the reaction is resealed by the recombinase. No external energy: released by ATP hydrolysis needed for DNA cleavage, & joining by these proteins. Site-Specific Recombinases Cleave & Rejoin DNA Using Covalent Protein–DNA Intermediate

12. Fig. 12-5. Covalent-intermediate mechanism used by Ser & Tyr recombinases. Here, OH group from an active-site serine is shown to attack phosphate: Introduce Single-strand break at site of recombination. The liberated OH group on broken DNA can then reattack the protein–DNA covalent bond to reverse this cleavage reaction : reseal DNA, & release protein. (Blue). Recombinase: Rec.

13. Serine Recombinases Introduce Double-Strand Breaks in DNA & Then Swap Strands to Promote Recombination During recombination, 4 single strands of DNA (two from each duplex) must be cleaved and then rejoined—now with a different partner strand— rearranged DNA. The serine recombinases cleave all 4 strands before strand exchange (Fig. 12-6). One molecule of the recombinase/ cleavage reaction involve R1, R2, R3, & R4 (Fig 12.6).

14. Fig. 12-6 Recombination by a ser recombinase. Each of 4 DNA strands cleaved within crossover region by one subunit of protein. These subunits are labeled R1, R2, R3, & R4. Cleavage of 2 individual strands of one duplex is staggered by 2 bases. This 2-bp region form hybrid duplex in recombinant products. The recombination sites (Darker colors).

15. Structure of Serine Recombinase –DNA Complex Indicate Subunits Rotate to Achieve Strand Exchange Interface b/w 2 cut DNA molecules is hydrophobic: therefore slippery so they will rotate with respect to each other. After 180 o rotation: there is some stability in structure due to +ve & -ve charges. Mechanism of recombination is (1) DNA cleavage: to form covalent enzyme–DNA intermediate; (2) an 180 o rotation of dimers in protein–DNA complex (3) attack of 3-OH DNA.

16. Tyrosine Recombinases Break & Rejoin One Pair of DNA Strands at a Time Tyrosine recombinases cleave & rejoin 2 DNA strands 1 st & only then cleave and rejoin the other two strands (Fig. 12-8). 4 molecules of the recombinase are needed. Cleavage occurs at the 1 st nucleotide of crossover region. Next, the right top strand from the top (gray) DNA molecule and the right top strand from bottom (red) DNA molecule “swap” partners. These 2 DNA strands are then joined: Now in Recombined configurations. “1 st -strand” exchange reaction generate branched DNA intermediate: Holliday junction . Once 1 st -strand exchange completed: 2 recombinase subunits (R2, R4) cleave bottom strand of each DNA molecule (Fig. 12-8c). These strands again switch partners & then joined by reversal of cleavage reaction. This “2 nd -strand” exchange reaction undo Holliday junction: DNA Product.

17. Fig. 12-8 Recombination by a Tyr Recombinase. Here R1, & R3 subunits cleave DNA in 1 st step (a); Protein becomes linked to cut DNA by 3` P-tyrosine bond. (b) Exchange of 1 st pair of strands occur: when 2 5`-OH groups at break sites each attack protein–DNA bond on other DNA molecule. (c, d) 2 nd -strand exchange occurs by the same mechanism: using R2, & R4 subunits.

18. Structures of Tyr Recombinases Bound to DNA Reveal Mechanism of DNA Exchange Cre Recombinase(site-specific DNA Recombinase): Enzyme encoded by phage P1, which functions to circularize linear phage genome (Recognize Lox P site in Phage). Cre acts, on lox sites. Cre –lox: e.g. of tyr recombinase family recombination Cre protein & lox sites are needed for complete recombination. Cre –lox: Recombination req 4 subunits of Cre , with each molecule bound to one binding site on the substrate DNA. Cre exists in 2 distinct conformations. Only in one of these conformation (Green subunits in fig) can Cre cleave, & rejoin DNA. Thus, only one pair of subunits is in the active conformation at a time. The pair of subunits in this active conformation switches as the reaction progresses.

19. Fig. 12-9 Mechanism of site-specific recombination by Cre recombinase. (a) Series of intermediate Cre –DNA structures: reflect sequential “one strand at a time” mechanism of exchange. In each of the panels: only 2-subunits colored in green are in the active conformation. Note that after 1 st -strand cleavage: Colors of the subunits switch as 2 nd pair of Cre subunits becomes active for recombination.

20. Cells & viruses use CSSR for a wide variety of biological functions. Many phage insert their DNA in host chromosome during infection using this recombination mechanism. In other cases, site-specific recombination is used to alter gene expression. For example, inversion of a DNA segment can allow two alternative genes to be expressed. SSR maintain structural integrity of circular DNA molecules. BIOLOGICAL ROLES OF SITE-SPECIFIC RECOMBINATION Mostly simple recombinase enough to bring 2 recombination site together for recombination. but some times other proteins also needed (Like architectural proteins, to bend DNA: bring 2 recombination sites together in synaptic complex). These proteins also give direction to recombination by preventing reverse reaction.

21. When bacteriophage λ infect host bacterium: series of regulatory events Dormant lysogenic cycle/Phage multiplication (Lytic Cycle/Growth). Lysogen requirement : Integration of phage DNA in host chromosome. When phage leave lysogenic state to replicate and make new phage particles: Excise its DNA from host chromosome. λ integrase protein ( λ Int ) catalyze recombination b/w 2 specific sites ( att /attachment sites). attP site is on phage DNA (P for phage), & attB site is in bacterial chromosome (B/Bacteria). λ Int : Tyr Recombinase. λ integration req accessory proteins: Req protein–DNA complex Assembly. Proteins: control the reaction (DNA integration, & DNA excision at right time). AttP & attB sites: Both carry a central core segment (30 bp). These core recombination sites: Each site consist of 2 λ Int -binding sites *& crossover region: Strand exchange occur. AttB only has core region: AttP is quite large (240 bp): Extra regions on both sides of core regions (Arms): Additional binding sites for proteins. Integrase Promote Integration and Excision of a viral Genome in Host-Cell Chromosome

22. Fig. 12-10. Recombination sites involved in λ integration & excision showing imp seq elements. C, C0, B, and B0 are core lInt -binding sites. The additional protein-binding sites are on attP and flank C, & C0 sites. These regions are called “arms”; the seq on the left : P arm, and those on the right: P0 arm. Small purple boxes labeled P1, P2, & P1 0 are arm lInt -binding sites. Sites marked H are integration host factor (IHF)-binding sites, & sites marked X are sites: Bind Xis . F is site bound by Fis (Another architectural protein not discussed further here. (Gray) The crossover regions. For clarity, lInt is not shown bound to the core sites. Note that not all protein-binding sites are filled during either integrative/ excisive recombination. After recombination, the P arm is part of attL , whereas the P0 arm become part of attR .

23. Fig. 12-11 Model for IHF bending DNA to bring DNA-binding sites together. The λ Int, & IHF-binding sites from P ´ arm of attP are shown. IHF binding to H ´ site bends the DNA to allow one molecule of lInt to bind both the P1 ´ & C ´ sites. The break in the DNA within H ´ site reflects nick that was present in DNA used for structural analysis of IHF–DNA complex.

24. Arms Having Protein binding sites: including additional sites bound by λ Int (labeled as P1, P2, and P0 (Fig. 12-10). λ Int u nusual protein: 2 Domains for seq -specific DNA binding: One domain binds to the arm recombinase recognition sites, 2 nd (Binds to core recognition sites)+ Architectural proteins. Integration req : attB , attP , λ Int , Architectural protein (AP) AP: Integration host factor (IHF): Seq -dep DNA-binding protein: introduce large bends ( > 160 o ) in DNA (Fig. 12-11). attP arms: Have 3 IHF-binding sites (H1, H2, H0: Fig. 12-10). IHF: Bring together λ Int sites on DNA arms (Where λ Int binds strongly), Sites present at central core ( λ Int binds only weakly). Bending of DNA (By IHF): λ Int to find weak core sites and to catalyze recombination. When recombination is complete: Circular Phage in host chromosome. 2 new hybrid sites generated at junction b/w phage & host DNA ( attL (left), attR (right). Both sites: Core region, 2 arm regions are now separated from each ther (P & P0 regions). Neither of 2 core regions in new arrangement competent to assemble active λ Int rec complex. DNA sites for assembly not in right place. λ Int catalyze recombination b/w 2 specific sites ( att B and P sites): Att P Site Arms

25. Additional architectural protein, this one phage encoded: essential for excisive recombination (For Excision) Xis (for “excise”) Xis binds to specific DNA seq & introduce bends in DNA. Xis function similar to IHF. Xis recognizes two seq motifs in one arm of attR (& also present in attP — marked X1 and X2 Fig. 12-10). Binding these sites introduces a large bend ( > 140 o ) Xis , lInt , & IHF together stimulate excision. Recombination b/w attP & attB =Integration Recombination b/w attR & attL =Excision Bacteriophage l Excision Require New DNA-Bending Protein You may notice that Xis binding sites: also present in attP . When Xis present in attP : Will bend DNA: Manner that IHF can not bring integrase site together (No integration/recombination b/w attP & attB site). So Xis only produced by phage when it enters the lytic cycle.

26. Salmonella Hin recombinase inverts two alternative forms of flagellin (H1, & H2 forms): protein component of the flagellar filament. Flagella are on the surface of the bacteria: common target for the immune system (Fig. 12-12). Hin (Ser recombinase, promote inversion using basic mechanism described above for this enzyme family. Invertible segment carries gene encoding Hin, as well as a promoter, which in one orientation is positioned to express the genes located outside of the invertible segment. Hin Recombinase Inverts a Segment of DNA Allowing Expression of Alternative Genes Fig. 12-12 Micrograph of bacteria (Salmonella) showing flagella. The color-enhanced scanning electron micrograph show Salmonella typhimurium (red) invading cultured human cells. The hair-like protrusions on bacteria are flagella

27. When promoter is in normal position: then genes fljB (H2 flagellin) and fljA (repressor of H1 flagelllin are expressed. When inverted these genes are not expressed and as repressor of the H1 flagellin is also not produced to its gene (located in other part of the genome) is expressed. Fig. 12-13 DNA inversion by Hin recombinase of Salmonella. Inversion of DNA segment between the hix sites flips a promoter (P) to give 2 alternative patterns of flagellin gene expression.

28. Hin recombination req seq in addition to hix sites. This short (60 bp) seq is an enhancer: Stimulate rate of recombination ~1000-fold. Enhancer function req bacterial Fis protein (Factor for inversion stimulation). Like IHF, Fis: Site-specific DNA-bending protein+ protein–protein contacts with Hin (Imp for recombination). Hin can actually assemble & pair hix recombination sites: Synaptic complex in absence of Fis. 3 DNA sites ( hixL , hixR , & enhancer) need to come together. Formation of this 3-way complex: Facilitated by - ive DNA supercoiling : Which stabilize association of the distant DNA sites. Another bacterial architectural protein, HU: Facilitate invertasome complex assembly. HU is a close structural homolog of IHF, yet in contrast to IHF, it binds DNA in seq- indep manner. Hin Recombination Requires a DNA Enhancer

29. Fig. 12-14 Complexes formed during Hin-catalyzed recombination. Hin protein alone recognizes & pairs 2 hix sites. When Fis protein is also present, 3-segment complex can form. This complex is called the invertasome & is most active complex for promoting recombination.

30. What is the biological rationale for control of that recombination occurs only b/w hix sites that are present on the same DNA molecule. And not that intermolecular DNA rearrangements: this process is not regulated and inversion happens randomly.

31. Site-specific recombination is critical to maintain circular DNA. Basic problem with circular DNA molecules: They sometimes form dimers and even higher multimeric form during Homologous recombination. A single homologous recombination event can generate one large circular chromosome. At cell division, this dimer pose major problem. Many circular DNA molecules carry seq recognized by site-specific recombinases called: Resolvases. But it must be in direction of monomerization & not again into dimers, this situation will be worst, so specific mechanisms for each recombinase are in place to enforse this directionlality . Recombinases Convert Multimeric Circular DNA Molecules into Monomers

32. Fig. 12-15 Circular DNA molecules can form multimers. Homologous recombination b/w 2 daughter DNA molecules during DN A replication generate dimeric chromosome (or plasmid). Site-specific recombination by the XerCD recombinase is then needed to generate monomeric DNA molecules needed for cell division.