GENETIC RECOMBINATION IN Escherichia coli AND BACTERIOPHAGES

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GENETIC RECOMBINATION IN Escherichia coli AND BACTERIOPHAGES : 

GENETIC RECOMBINATION IN Escherichia coli AND BACTERIOPHAGES MICROBIOLOGY

INTRODUCTION : 

INTRODUCTION LINKAGE : Genes that are located in the same chromosome tend to stay together during inheritance. This tendency is called linkage. They belong to the same linkage group. The phenomenon of inheritance of linked genes in same linkage group is called linkage. A LINKAGE GROUP includes all loci that can be connected ( directly or indirectly ) by linkage relationships; equivalent to a chromosome. Linkage are of TWO types : COMPLETE LINKAGE INCOMPLETE LINKAGE

Slide 3: 

The CROSSING OVER is a process that produces new combinations ( RECOMBINATION ) of genes by interchanging of corresponding segments between non-sister chromatids of homologous chromosome. Types of crossing over: Somatic or mitotic crossing over Germinal or meiotic crossing over

RECOMBINATION IN E. coli : PATHWAYS AND PROTEINS : 

RECOMBINATION IN E. coli : PATHWAYS AND PROTEINS RECOMBINATION PATHWAYS IN E. Coli : The results of genetic analyses indicate that there are three distinct recombination pathways in E. coli:

THE RecBCD PATHWAY : 

THE RecBCD PATHWAY This is the major pathway of recombination in E. coli. In addition to the RecB, RecC and RecD proteins, it requires RecA as well as SSB, Pol I, DNA ligase and DNA gyrase.

THE RecF PATHWAY : 

THE RecF PATHWAY This pathway is the means by which plasmids recombine with one another. RecA is required as well as a number of other proteins (RecJ - an exonuclease, RecN, RecO, RecQ - a helicase) whose exact function or role is not known. "The RecF pathway of recombination is thought to involve interactions of RecF and its associated proteins on single stranded DNA gaps left by discontinuous DNA synthesis and conjugation to initiate recombination."

THE RecE PATHWAY : 

THE RecE PATHWAY RecE encodes exonuclease VIII(??), a 5' -> 3' exonuclease that is specific for dsDNA. This pathway requires many of the same gene products as the RecF pathway.

Recombination Proteins in E. coli : 

Recombination Proteins in E. coli The characterization of the structures and biochemical function of a number of the key proteins required for recombination is now helping us to understand details of the mechanism of recombination. The most important are RecA, RecBCD, RuvA, RuvB and RuvC.

RecA : 

RecA The RecA protein is a multifunctional powerhouse! It has strand-exchange, ATPase and co-protease activities all packed into a compact 352 amino-acid, 38 kDa structure. It is required for all recombination pathways in E. coli. The RecA protein will bind cooperatively to a ssDNA molecule with each monomer of RecA binding to a span of 4-6 nucleotides. Assembly of the nucleoprotein complex proceeds in a 5' -> 3' direction. The complex is both fairly stable (half-life is 30 min) and is the active species that will promote strand exchange.

Slide 10: 

THE STRAND EXCHANGE REACTION PROBABLY INVOLVES THE FOLLOWING STEPS: RecA binds to the ssDNA partner. The two molecules are aligned possible through the formation of a triple-stranded intermediate. Displacement of one of the old strands. This requires concurrent migration of the RecA nucleoprotein filament along the molecule - which proceeds in one direction only (5' -> 3') - and consequent winding/unwinding. ATP hydrolysis takes place during this step. RecA will promote strand exchange between DNA molecules as long as the following conditions apply: One of the two molecules must have a ssDNA region to which RecA can bind. The two molecules must share a region of homologous (i.e. nearly identical) DNA sequence - a minimum of 50 bp is required. There must be a free end within this region of homology which can initiate the strand exchange.

Slide 11: 

The RecA protein monomer structure is shown above. Monomers associate with one another via the N-terminal alpha helix which interacts with the adjacent monomer – forming a molecular conga line, if you will, with one helix on the shoulder of the molecule in front of it! The RecA filament that forms is helical with a pitch of 82.7 Å and it consists of 6 monomer units per turn.

Rec BCD : 

Rec BCD The recB, recC & recD genes code for the three subunits of the Rec BCD enzyme which has five activities: exonuclease V; a helicase activity; an endonuclease activity; an ATPase activity; and, an ssDNA exonuclease activity. RecBCD can unwind DNA faster than it rewinds. Thus as it travels along a DNA molecule, it generates ssDNA loops. When the enzyme encounters a Chi sequence, it cleaves the DNA nearby. This generates a ssDNA region that can serve (along with RecA) to initiate strand exchange and a recombination reaction. Chi sites were originally characterized because they are Crossover Hotspot Instigators. The site has the sequence GCTGGTGG. RecBCD cleaves several nucleotides to the 3' of this site.

RuvA : 

RuvA RuvA is a small protein whose function is to recognize a Holliday junction thereby assisting the RuvB helicase to promote branch migration. The crystal structure of the E. coli RuvA protein was solved at a resolution of 1.9 Å. (See The protein forms a tetramer in an unusual manner - though one that is ideally suited to its function. "Four monomers of RuvA are related by fourfold symmetry in a manner reminiscent of a four-petaled flower. The four DNA duplex arms of a Holliday junction can be modeled in a square planar configuration and docked into grooves on the concave surface of the protein around a central pin that may facilitate strand separation during the migration reaction."  The RuvA protein is 203 amino acids in length but only 190 of them could be assigned in the crystal structure. Most of the missing assignments represent amino acids in a flexible part of the protein.

Slide 14: 

It shows a single subunit of the RuvA tetramer. The flexible linker region is depicted as a dashed yellow line. It shows the "four-petalled" symmetry of the tetramer.

Slide 15: 

Fig. 3b (left) and Fig. 3c (center) show the charge surface of the tetramer complex in blue (positively charged) or red (negatively charged) associated with a Holliday intermediate (backbone strands only are shown). Fig. 3d (right) is a space-filling representation. Notice how compact the central region is -- very little distortion of the DNA double helices is required to form this structure

Slide 16: 

The RuvA-Holliday junction complexes have the predicted structure with a four-fold symmetry. The DNA bound at the junction is not flat but slightly concave to maximize the contact between the DNA and the protein and, as a result, the axes of the four helices are inclined at about 10 degrees. In the centre of the complex, eight residues corresponding to Glu-55 and Asp-56 from each of the four subunits, form an acidic central pin which may repel the DNA backbone away from the centre of the junction thereby, opening up the centre. These refined structures have also shown that there are only 4 unpaired bases in the centre of the complex. This is a lot fewer than found for typical helicases.

RuvB : 

RuvB The RuvB protein is a helicase that catalyzes branch migration of Holliday junctions. By itself it cannot bind to DNA efficiently. It functions in combination with RuvA. Like other helicases, RuvB functions as a hexamer; but, unlike other helicases, RuvB encloses double-stranded DNA not ssDNA. Electron microscopy has shown that RuvB is a heptamer in solution and that it converts to a hexamer ring when it binds to DNA. Electron microscopy has also shown that the two hexamer rings of RuvB lie contacting RuvA on the two opposite sides of a RuvAB-Holliday junction complex. A hypothetical hexamer model of RuvB compares well with the images generated by electron microscopy [figure]. See also RuvB protein bound to ds linear DNA.

RuvC : 

RuvC The RuvC protein resolves the Holliday intermediate. It functions as a dimer to cleave two of the four strands that make up the central part of the intermediate. Since binding is symmetrical, RuvC can bind to the Holliday intermediate in two equally likely ways. Hence, Holliday intermediates can be resolved in two different, but equally likely, ways. The interaction of RuvC with Holliday junction is shown. RuvC does have some sequence specificity. It cleaves DNA at the 3'-side of thymidine, preferentially at the consensus 5'-A/TTT|C/G -3' where | indicates the site of cleavage

Slide 19: 

RuvC dimer in association with a Holliday junction.

Slide 23: 

Proposed function of RecBCD and RecA in homologous recombination in E. coli (figure from Kowaczykowski, 2000). RecBCD enzyme binds to a blunt-end DNA from a double-stranded DNA break. It unwinds the dsDNA and prefereintially degrades the 3’-terminating strand (top strand). Interaction with χ results in attenuation of the 3’ → 5’ nuclease activity, the activation of a weaker 5’ → 3’ nuclease activity, and the facilitated loading of RecA protein onto the χ-containing ssDNA. The resulting RecA/ssDNA filament can then invade homologous dsDNA.

ENZYMES INVOLVED IN HOMOLOGOUS RECOMBINATION : 

ENZYMES INVOLVED IN HOMOLOGOUS RECOMBINATION

Site-specific recombination : 

Site-specific recombination In site-specific-recombination, DNA strand exchange takes place between segments possessing only a limited degree of sequence homology (Kolb 2002; Coates et al., 2005). Site-specific recombinases perform rearrangements of DNA segments by recognising and binding to short DNA sequences (sites), at which they: cleave the DNA backbone, exchange the two DNA helices involved and rejoin the DNA strands. While in some site-specific recombination systems having just a recombinase enzyme together with the recombination sites is perfectly adequate to be able to perform all these reactions, in some other systems a number of accessory proteins and accessory sites are also needed. The 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 e.g. attP and attB of λ integrase (Landy 1989)(see lambda phage).

Slide 27: 

The reaction catalysed by the recombinase may lead to the excision of the DNA segment between flanked by the two sites, but also to the integration or inversion of the orientation of the flanked DNA segment. What the outcome of the reaction will be, is dictated mainly by the relative location and the orientation of sites that are to be recombined, but also by the innate specificity of the site-specific system in question. Excisions and inversions occur if the recombination takes place between two sites that are found on the same molecule (intramolecular recombination), and if the sites are in the same (direct repeat) or in an opposite orientation (inverted repeat), respectively. Insertions on the other hand take place if the recombination occurs on sites that are situated on two different DNA molecules (intermolecular recombination), provided that at least one of these molecules is circular. Most site-specific systems are highly specialised catalysing only one of these different types of reaction and have evolved to ignore the sites that are in the ‘wrong’ orientation. In nature site-specific recombination systems are highly specific, fast and efficient, even when faced with complex eukaryotic genomes (Bode et al., 2000; Sauer 1998). As such, they are employed in number of processes such as: bacterial genome replication, differentiation and pathogenesis, movement of genetic elements such as transposons, plasmids, phages and integrons (Nash 1996) and present an attractive starting material for development of potential genetic engineering .

Classification of site-specific recombinases: tyrosine vs. serine recombinases : 

Classification of site-specific recombinases: tyrosine vs. serine recombinases Based on amino acid sequence homology and mechanistic relatedness most site-specific recombinases are grouped into one of two families: the tyrosine recombinase family or the serine recombinase family. The names stem from the conserved nucleophilic amino acid residue that they use to attack the DNA and which becomes covalently linked to it during strand exchange. Serine recombinase family is also sometimes known as resolvase/invertase family, while tyrosine recombinases are known as the integrase family, which reflects the types of reaction that most known members in each family have evolved to catalyse. Typical examples of tyrosine recombinases are the well known enzymes such as Cre (from the P1 phage), FLP (from yeast S. cerevisiae) and λ integrase (lambda phage) while famous serine recombinases include enzymes such as: gamma-delta resolvase (from the Tn1000 transposon), Tn3 resolvase (from the Tn3 transposon) and [[φC31 integrase]] (from the φC31 phage) (Nash 1996; Stark and Boocock 1995). Although the individual members of the two recombinase families can perform reactions with same practical outcomes, the two families are unrelated to each other, having different protein structures and reaction mechanisms. Unlike tyrosine recombinases, serine recombinases are highly modular as was first hinted by the biochemnical studies (Abdel-Meguid et al., 1984) and later shown by crystallographic structures (Yang and Steitz, 1995; Li et al., 2005); a fact which could prove useful when attempting to reengineer these proteins as tools for genetic manipulation.

Mechanism and the chemistry of the site-specific DNA recombination reaction : 

Mechanism and the chemistry of the site-specific DNA recombination reaction Recombination between two DNA sites begins by the recognition and binding of these sites by the recombinase protein. This is followed by the synapsis i.e. bringing the sites together to form the synaptic complex. It is within this synaptic complex that the strand exchange takes place, as the DNA is cleaved and rejoined by controlled transesterification reactions. During strand exchange, the DNA cut at fixed points within crossover region of the site releases a deoxyribose hydroxyl group, while recombinase protein forms a transient covalent bond to a DNA backbone phosphate. This phosphodiester bond between 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. The entire process therefore goes through without the need for external energy rich cofactors such as ATP. As stated previously, the recombination sites are slightly asymmetric, which allows the enzyme to tell apart the left and right ends of the site. When generating products left ends are always joined to the right ends of their partner sites and vice versa. This causes the recombination sites to be reconstituted in the recombination products. Joining of left ends to left or right to right is avoided due to the asymmetric “overlap” sequence between the staggered points of top and bottom strand exchange. Left-left or right-right half-site recombinants would contain mismatched base pairs (Stark and Boocock 1995). Although the basic chemical reaction is the same for both tyrosine and serine recombinases there are marked differences.

Slide 30: 

Tyrosine recombinases, such as Cre or FLP, cleave one DNA strand at the time at points that are staggered by 6-8bp, linking 3’ end of DNA to the hydroxyl group of the tyrosine nucleophile (Van Duyne 2002). Strand exchange than proceeds via a crossed strand intermediate analogous to the Holliday junction (Holliday 1964; Grainge and Jayaram 1999) in which only one pair of strands has been exchanged. Conversely, serine recombinases like gamma-delta and Tn3 resolvase cut all four DNA strands simultaneously at points that are staggered by 2bp (Stark et al., 1992). During cleavage protein-DNA bond is formed via transesterification reaction in which a phosphodiester bond is replaced by a phosphoserine bond between a 5’ phosphate at the cleavage site and hydroxyl group of the conserved serine residue (S10) in resolvase (Reed and Grindley 1981; Reed and Moser 1984). It is still not entirely clear how the strand exchange occurs after the DNA has been cleaved. However, it has been shown that the strands are exchanged while covalently linked to the protein with a resulting net rotation of 180° (Stark et al., 1989; Stark and Boocock 1994). Two current models can account for this, namely the subunit rotation model and the domain swapping model (Sarkis et al., 2001). In both of these models DNA duplexes are situated outside of the protein complex, and large movement of protein is needed to achieve the strand exchange. This is in stark contrast to the mechanism employed by the tyrosine recombinases.

BIBLIOGRAPHY : 

BIBLIOGRAPHY Genes VIII Principles of microbiology - Ronald M. Atlas. Microbiology - Lansing M. Prescott John P. Harley Donald A. Klein Biotechnology - V. Kumaresan Google Wikipedia

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