Mechanisms Of Conservative And Replicative Transposition Biology Essay
Bacteriophage Mu is a temperate phage which adopts heterotaxy tract in its life rhythm. Mu has the capableness to incorporate into legion sites in host Escherichia coli genome and cause mutants due to its insertional activation. Mu transposes via two major tracts ; conservative and replicative heterotaxy though the molecular switch between the two mechanisms remain unknown. This reappraisal will concentrate on the comparings between replicative and conservative heterotaxy. The first portion will discourse the similarities between the two mechanisms ; donor DNA cleavage measure and strand transportation measure which involves nucleophilic onslaughts, bring forthing single-strand dents in Mu DNA and fall ining it to aim DNA via one-step transesterification mechanism. The latter portion will concentrate on the different features in each heterotaxy mechanism ; in replicative heterotaxy, the terminal merchandise is duplicate of jumping gene transcript in both mark and host DNA while in conservative heterotaxy, a simple interpolation of jumping gene is produced in the mark DNA.
1. Features of bacteriophage Mu
Bacteriophage, derived from the Grecian word phagein, literally means “ to eat ” . Bacteriophage Mu was named as such ( happen out who did ) due its nature of infecting and bring oning high degrees of mutant in host bacteriums Escherichia coli. , therefore the name “ Mu ” for mutator. The double nature of Mu – jumping gene and virus – has made it as the archetypical theoretical account of analyzing phage genetic sciences. Bacteriophage Mu is a temperate phage of E. coli which employs the heterotaxy mechanism in its life rhythm. Transposition can either be conservative ( striking the jumping gene and infixing it into bacterial chromosome ) or replicative ( transposon transcripts are produced in both jumping gene and bacterial chromosome ) . Both mechanisms will be discussed extensively later in this article. Unlike the phage I» , interpolation of Mu genome into the mark site returns in a randomly mode which makes it an first-class mutator.
Fig. 1: The life rhythm of bacteriophage Mu ( 5 ) .
The life rhythm of phage Mu is shown schematically in Fig. 1 above. Bacteriophage Mu infect susceptible host cell by surface assimilation and so, injects its additive viral genome. Once inside the host cell, the additive genome does non circularized ( 4,5,19 ) , unlike in phage I» . In either instance of lytic or lysogenic stage, Mu integrates its Deoxyribonucleic acid into the host genome via conservative heterotaxy ( 16,19 ) . This is observed otherwise in phage I» where the infecting phage DNA will be integrated into host genome merely during lysogenisation ( 19 ) . An enzyme called transposase, encoded by MuA cistron in the phage genome, is perfectly important to transport out this conservative heterotaxy measure. Phage DNA is inserted at multiple sites in a bacterial genome which lead to the premise that the interpolation occur by a random mode ( 8 ) . However, there are several factors that influence mark site choice such as MuA protein efficiency and heterotaxy unsusceptibility ( 15 ) .
After integrating, Mu normally adopts a quiescent prophage life style ( lysogenic stage ) . The penchant between lysogenic and lytic stage in Mu life rhythm is dependent on its stableness in the lysogen and lysogenic repressers. However, lysogens of Mu phage sometimes enter the lytic stage though this is a rare event. When induced, normally by utilizing temperature-sensitive represser mutations of phage Mu and capable it at 42EsC, the lysogen will come in lytic rhythm. When the lysogenic represser is inactivated, Mu transposes via replicative heterotaxy, bring forthing transcripts of phage genome which will be packaged into new virions. The virions so lyse the host cell and infect new hosts. Bacteriophage Mu virions comprised of icosahedral caput ( diameter 54nm ) , a baseplate, a contractile tail and six short tail fibers ( 5 ) .
Fig. 2: Simplified sketch exemplifying packaging of Mu genome. Typical length of phage Mu DNA is about 37kb long. Extra 2 kilobit of host DNA is incorporated during DNA packaging which is shown as flanking each terminal of the integrated Mu genome, with most of it at the right terminal. Unique sequences of host Deoxyribonucleic acid and at the right terminal of the packaged Deoxyribonucleic acid is dependent on induction site of packaging in the host DNA ( 24 ) .
Fig. 3: Physical and familial map of bacteriophage Mu. Solid black lines represent Mu DNA while the boxes at the two terminals indicate flanking host DNA sequences. Mu cistrons ( indicated in block letters ) and their corresponding translational merchandises are as indicated ( 19 ) .
A typical size of wild-type phage Mu DNA is about 37.5 kilobits, nevertheless each phage mirid bug can suit up to 39 kilobits long. Bacteriophage genome has a political action committee site which serves as the get downing point in packaging of the phage DNA, located within attL ( 5 ) . The induction cleavage by phage enzyme terminase occurs upstream of the phage political action committee site, which includes host sequence of about 50-150bp flanking the left terminal. Second cleavage initiated when a complete filling of mirid bug is achieved, which includes 0.5 kilobit to 2 kilobit of host sequence flanking the right terminal ( 1 ) . Familial and physical map of phage Mu is illustrated in Fig. 3. Bacteriophage Mu utilizes ‘headful ‘ mechanism scheme, which confer variable lengths of host DNA flanking the left terminals of Mu DNA depending on the induction site of genome packaging ( Fig. 2 ) .
2. Transposition mechanism
( Tocopherol )
( D )
( C )
( B )
( A )
Fig. 4: Manners of bacteriophage Mu heterotaxy. ( A ) , ( B ) and ( C ) are the common stairss in both conservative and replicative heterotaxy of phage Mu. In conservative and replicative heterotaxy, phage Mu will follow-up measure ( D ) and ( E ) severally. Curved arrows indicate nucleophile onslaught, reassigning the 3′-OH terminals to the staggered 5′-phosphate terminals of mark DNA. Dentate lines ( XXXX ) indicate mark DNA sequences which are duplicated during heterotaxy ( 16 ) .
Numerous in vitro surveies have been conducted to analyze the mechanism of heterotaxy, and normally mini-Mu elements are used. A minimum Mu component consists of a selectable cistron, a plasmid reproduction beginning and indispensable Mu ends ( 2 ) . The mechanism of heterotaxy is discussed in regard to an in vitro system from this point onwards unless stated otherwise. Following treatment on heterotaxy mechanism are based on Shapiro theoretical account ( 22 ) as it has been widely accepted as the ‘golden ‘ theoretical account in this field.
The current known manners of heterotaxy is divided into two: non-replicative ( conservative ) and replicative heterotaxy. Both schemes utilize the same mechanism up to indicate ( Fig. 4C ) where each scheme employs different mechanism, bring forthing different terminal merchandises. A simple interpolation of jumping gene is generated in mark DNA by conservative heterotaxy ( Fig. 4D ) while two transcripts of jumping gene formed in both giver and mark Deoxyribonucleic acid by replicative heterotaxy ( Fig. 4E ) . Indicate A to C are considered as the similar characteristics in both conservative and replicative heterotaxy while point D and E is the differentiation between the two manners of heterotaxy. Therefore, mechanisms involved in point A, B and C are discussed in context of both replicative and conservative heterotaxy, which comprises of DNA cleavage measure and strand transportation measure. Consecutive phases of both cleavage and strand transportation stairss are illustrated in Fig. 4.
2.1 Donor DNA cleavage measure
Two critical chemical stairss in both heterotaxy tracts are donor DNA cleavage measure and DNA strand reassign measure ( 5,8 ) . The giver DNA cleavage measure is initiated when H2O molecules within an active site act as nucleophiles, and onslaught phosphodiester bond in DNA anchor at each of the transposon terminal ( 4,5 ) . The cleavage measure involves a direct hydrolysis of phosphodiester bond by H2O, and non by covalent enzyme-DNA intermediate ( 17 ) . The phosphodiester bond is cleaved at the flanking host-transposon DNA boundary. 3′-hydroxyl ( OH ) ends of the Mu DNA are exposed at the terminal of the cleavage measure. Strand transportation consequences in merger of mark and giver DNA, which forms an intermediate molecule ( 8 ) . The procedure ( simplified in Fig. 4C ) follows the Shapiro theoretical account ( 22 ) .
Bacteriophage-encoded proteins, MuA protein ( transposase ) and MuB protein ( ATPase ) are required for heterotaxy. Other demands to guarantee efficiency of heterotaxy are accessary proteins such as host-encoded DNA bending proteins called hydroxyurea ( HU ) and integrating host factor ( IHF ) ( 8 ) . The upside-down repetitions at the terminal of giver DNA, and mark sequence on bacterial chromosome are besides of import in heterotaxy mechanism. The assembly of higher order protein-DNA composites called transposome has been identified by in vitro surveies ( 6 ) .
A three-site synaptic composite called the LER complex comprising right and left terminals of Mu and transpositional foil, was formed in the beginning of heterotaxy in vitro ( 23 ) . MuA protein binds to MuA adhering site at the terminals of Mu DNA as monomer, and later map as tetramer of MuA ( transposase ) . Host IHF and HU protein were found to help in formation and stabilization of LER composite.
The LER composite is comparatively unstable and so, is quickly converted into stable synaptic composite ( SSC ) , besides known as type 0 complex ( 17 ) . This is the critical checkpoint before any chemical reaction is carried out as it is the rate-limiting measure of cleavage reaction ( 6 ) . A stable synapse between tetramer of MuA and the two terminals of Mu DNA is made but no cleavage is initiated yet at this point. Nonetheless, the active site is structurally occupied to the part around the scissile phosphate while the flanking Deoxyribonucleic acid are destabilized upon formation of the SSC composite ( 6 ) . In add-on to formation of a stable synapse, the Mu ends demands to be properly-oriented, a super coiled DNA topology, and accessary DNA sites are besides of import to continue to the following measure. Formation of SSC normally is ephemeral in presence of Mg2+ but can be accumulated in presence of suited bivalent cations such as Ca2+ , which promotes the formation of SSC ( 8,17 ) .
Following, SSC is converted into a type 1 transposome composite, besides called as cleaved giver composite ( CDC ) ( 9 ) . The 3 ‘ terminals of Mu DNA are nicked in presence of Mg2+ . Two fractional monetary units of MuA tetramer, that are associated with the sites that undergo cleavage, assemble in trans agreement which favours the strand transportation reaction ( 5 ) . The formation of CDC can so be thought as the consequence of giver DNA cleavage measure. Type 1 transposome complex exhibits greater stableness than the type 0 complex though MuA forms structural and functional nucleus in both transposome composite ( 6 ) . In add-on of stably bound tetramer of MuA proteins, there are slackly associated MuA proteins present in the CDC every bit good. In absence of MuB protein, MuA tetramer is unable to advance strand transportation reaction unless these excess MuA proteins are present. MuB protein is an ATP-dependent DNA-binding protein, which besides acts as an allosteric activator of Mu transposase ( MuA proteins ) ( 21 ) . Transposition can still continue in absence of MuB proteins, but MuA protein by itself is merely 1 % efficient ( 3 ) .
2.2 Strand transportation measure
A trademark of this measure is the formation of strand transportation composite ( STC ) , besides known as type 2 transposome composite. The terminal merchandise of STC is formation of a bifurcate molecule ( Shapiro intermediate ) which is characterized by a covalent interaction between giver DNA and aim DNA via 5bp single-stranded spreads and its I? construction ( 22 ) . MuB protein foremost captures a mark molecule and convey it to the locality of the transposome composite, organizing a TC composite ( 6 ) . Formation of TC composites quickly undergo one-step transesterification reaction, which is the rate-limiting measure in the strand transportation measure. Interestingly, recruiting of mark molecules by MuB proteins and formation of TC composites can happen at several clip point during the reaction tract ( 6 ) . This is a peculiarly efficient measure to maximise heterotaxy potency as it would rush up rate of strand transportations during heterotaxy.
The free 3′-OH terminals produced from the cleavage measure act as nucleophile and attack phosphates of mark Deoxyribonucleic acid at the 5 ‘ terminals. 5-nucleotides long beginning dents are made in the mark DNA, bring forthing a staggered agreement ( 3 ) . At this phase, the MuA proteins ( transposase ) are still tightly bound to the branched molecule with individual stranded spreads. This pose an obstructor for the assembly of reproduction fork by host reproduction factors. The construction of the bifurcate molecule is simplified in ( C ) of Fig. 4.
The forming of this intermediate molecule serves as the critical point which distinguish between conservative and replicative heterotaxy. A widely accepted theoretical account is that the resolution of this co-integrate molecule by a particular resolvase complex leads to duplicate transcripts of jumping gene being made in both giver and mark site ( REFerence ) . This is by definition, a replicative heterotaxy tract. Therefore, the strand transportation composite is destabilized and disassembled by a system of eight E. coli host molecular proteins ( DnaB helicase, DnaC protein, DnaG primase, DNA polymerase II, single-strand binding protein, DNA gyrase, DNA polymerase I and DNA ligase ) and molecular chaperon called ClpX, bring forthing cointegrates ( 13 ) .
This passage from transposome composite to a replisome consequences in duplicate of 5-bp mark DNA sequences flanking both terminals of Mu DNA. Alternatively, if the bacteriophage Mu is to come in the conservative tract, the co-integrate molecule is repaired or processed without executing Mu DNA reproduction. The terminal merchandise of STC in a conservative heterotaxy is a simple interpolation of individual mini-Mu component inserted into the mark DNA ( 8 ) . However, the mechanism of this theoretical account is ill understood.
Fig. 5: Transposome composites involved during DNA cleavage composite and DNA strand transportation. ( A ) A plasmid ( grey line ) bearing donor mini-Mu component ( black line ) Deoxyribonucleic acid in the in vitro system is negatively coiled. ( B ) In presence of host HU protein, Mu A protein bind to the two terminals of Mu DNA organizing a stable synaptic composite ( non shown ) . Assembly of MuA tetramer produces a dent at each terminals of Mu DNA, making a cleaved giver composite ( CDC ) . ( C ) Nicked 3 ‘ terminals of Mu DNA are joined together to aim DNA in presence of MuB protein organizing a strand transportation composite ( STC ) . MuA tetramer is still tightly bound to the Mu ends in the STC. ( D ) In replicative heterotaxy, a cointegrate molecule is produced when reproduction of mark DNA initiated from the 3 ‘ Mu terminals by host reproduction machinery ( 13 ) .
3. Replicative heterotaxy
Replicative heterotaxy was foremost suggested by Ljungquist and Bukhari ( 1977 ) to happen in situ after initiation of lysogens, which means that the Mu prophage was non excised from host chromosome during heterotaxy ( 14 ) . The lysogens were digested with limitation enzymes which cleaves both host and Mu DNA at specific limitation sites. Two of the fragments from the limitation digests contain both host and Mu DNA, which corresponds to junctions between host and prophage Deoxyribonucleic acid, proposing that prophage Deoxyribonucleic acid is replicated in situ of host chromosome ( 19 ) . Several familial and biochemical anticipations made in the Shapiro theoretical account have been demonstrated in both in vivo and in vitro surveies, therefore this theoretical account is accepted as a plausible mechanism to explicate heterotaxy in phage Mu.
Numerous techniques have been done to analyze the way of reproduction of Mu DNA during heterotaxy. Consequences obtained by tempering of Okazaki fragments to detached strands of Mu DNA shows that more than 80 % of Mu molecules replication proceed from left to right terminal ( 11,19 ) . Electron microscopical observation of mini-Mu component shows that retroflexing molecules in vitro replicate from both terminals in ‘equal chance ‘ ( 11,19 ) . Reproduction of Mu DNA is accepted to be preponderantly unidirectional, that is from left towards the right terminal ( 20 ) . Intramolecular reproduction tract can ensue in inversion, omission, and simple interpolation while intermolecular events can bring forth co-integrate molecules ( 19 ) . In the instance of Mu heterotaxy, formation of co-integrate molecule demands to be resolved in order to bring forth two replicons ; one molecule contains jumping gene and mark DNA while another molecule contains jumping gene and giver DNA ( 10 ) .
4. Conservative heterotaxy
The chief characteristic of conservative heterotaxy is that phage DNA is non replicated prior to integrating. Upon infection of a susceptible host cell ( normally E. coli ) , Mu employs conservative, or besides called non-replicative heterotaxy to reassign its genome to the mark site. As discussed earlier, conservative heterotaxy tract follows individual strand dents at the 3 ‘ terminals of Mu DNA, of which the exposed 3’-OH terminals articulation to the staggered cut mark DNA at the 5’ends organizing a co-integrate molecule. The co-integrate or alleged Shapiro intermediate is repaired and generates a simple interpolation in the mark DNA though the mechanism is still ill understood.
Shapiro theoretical account emphasized on single-stranded dents at Mu terminals, connection of Mu to a staggered double-strand interruption in mark DNA, formation of an intermediate molecule, and casting of heterogenous of old host DNA sequences after ligation in conservative tract ( 22 ) . On the other manus, Morisato and Kleckner ( 1984 ) proposed a different mechanism based on consequences with Tn10 heterotaxy. Their theoretical account is double-stranded cleavages at the jumping gene ends bring forthing an excised jumping gene, which so circularizes via ligation on one of the strands ( 18 ) . It predicts casting of host sequences from the Mu DNA ends before ligation into the new mark DNA. Study of Mu heterotaxy utilizing plasmid substrates in vitro produced consequences in favor of the Shapiro theoretical account, and therefore this theoretical account has been widely accepted and used in surveies.
Fig. 6: A theoretical account of conservative heterotaxy which utilizes double-strand cleavages during integrating. ( A ) Transposase bind to the upside-down repetitions at Mu-host boundary sites and cleaves off the jumping gene off. ( B ) Transposase made a staggered cut at mark sequence of which exposed 3′-OH terminals of jumping gene onslaughts 5′-phosphate terminals of the host ( non shown ) . The jumping gene so joins to the host sequence. Duplicated mark sequence of 5-bp are completed by host reproduction machinery ( 7 ) .
The argument on single-strand or double-strand cleavage nevertheless does non stop at that place. If phage Mu were to use the Shapiro theoretical account of heterotaxy during integrating ( the well-established cointegrate mechanism ) , the flanking host sequences would stay bound to Mu ends. This would clearly present a job as subsequent target-primed reproduction of the additive integrant would non work, or merely interrupt the chromosome ( 1 ) . Obviously, consequences from in vitro experiments are against this as the heterotaxy terminal merchandises contain jumping gene, proposing a complete heterotaxy procedure have been accomplished. So, does the infecting Mu DNA use the Shapiro theoretical account where the cointegrate molecule gets processed and repaired, prior to reproduction at the flanking sequence? Or does it follow a cut-and-paste mechanism where both strands of Mu DNA gets cleaved off from the flanking host DNA sequence ( as illustrated in Fig. 6 ) , where no cointegrate molecule is generated, which finally means, there is no demand for resoluteness by reproduction?
An in vitro experiment was done by Au et Al. ( 2006 ) to detect the destiny of flanking host Deoxyribonucleic acid sequences upon phage Mu infection. Specific markers specific to the infecting phage Mu DNA every bit good as the giver host ( lacZ/proB ) were used. These markers were acquired from the host in which the phage had been propagated but absent in the host being infected ( 1 ) . Upon infection of plasmids by bacteriophage Mu, signal for flanking sequences and Mu DNA were detected in the chromosome at the same clip point ( about at infinitesimal 8 ) , which correspond to the integrating clip point of Mu. Subsequent look of lacZ and proB were detected maximally at minute 15, significantly reduced at minute 30 and by minute 50, look were halted ( 1 ) . Maximal look at minute 15 most likely corresponds to culminate of integrating of the infecting phage population. These findings strongly suggest that flanking sequences get integrated together with Mu DNA into the new mark site and are later, removed by a particular mechanism ( which explained the undetectable look at minute 50 ) . This so proves that infecting phage Mu employs an alternate cointegrate mechanism ( besides called as nick-join-process mechanism ) in conservative heterotaxy tract, where the Mu DNA undergo single-strand dents, joins to the mark DNA, and repaired before reproduction of the 5-bp spread left by the flanking sequence ( 1 ) . The mechanism of remotion and fix of host flanking sequence nevertheless, remains equivocal.
Double nature of bacteriophage Mu, a permutable component and a virus, is surely interesting but what is more absorbing is that it utilizes both replicative and non-replicative heterotaxy throughout its life rhythm. The former mechanism produces a jumping gene transcript in both giver and mark DNA while the latter normally generates a simple interpolation of jumping gene in the mark DNA, go forthing a spread in the host DNA which most probably will acquire degraded.
In the early phases, both replicative and conservative heterotaxy tract portion a similar mechanism. Regardless of the heterotaxy tract, infecting Mu DNA during the first unit of ammunition of infection will incorporate its DNA into the mark chromosome via two critical stairss ; donor DNA cleavage measure and strand transportation measure. Mu uses a phosphoryl transportation affecting nucleophilic onslaughts of H2O on phosphodiester bonds of Mu DNA, bring forthing single-strand dents. A 2nd nucleophilic onslaught by open 3′-ends of Mu DNA on 5′-ends of mark phosphodiester bonds, which so joins the Mu DNA to aim DNA via one-step transesterification mechanism. A series of transposome composites are formed throughout these procedures including Mu-encoded MuA proteins ( transposase ) and MuB proteins ( ATPase ) . A cointegrate is produced in both tracts but in replicative heterotaxy, this intermediate molecule is resolved bring forthing two replicons with jumping gene transcript in each molecule. In conservative heterotaxy, the cointegrate is repaired bring forthing a simple interpolation in the mark DNA. Hence, it is more accurate to call conservative heterotaxy as ‘nick-join-process ‘ instead than the conventional ‘cut-and-paste ‘ mechanism as the latter suggest double-strand dents at the transposon terminal, which has been proven inaccurate by in vitro experiments. Both heterotaxy tracts have been compared extensively in this reappraisal but much of functional nucleus of the mechanisms remain to be understood.