Mechanisms Of Conservative And Replicative Transposition Biology Essay

Mechanisms Of Conservative And Replicative Transposition Biology Essay bacterio phage Mu is a clement phage which adopts turn around roadway in its heart cycle. Mu has the capability to desegregate into numerous invests in force Escherichia coli genome and cause mutations due to its foundingal activation. Mu trans awards via 2 study pathways justifiedly and replicative turn around though the molecular switch between the devil chemical utensils remain unknown. This review testament focus on the comparisons between replicative and conservative policy change. The prime(prenominal) representative pull up stakes discuss the similarities between the deuce machines bestower desoxyribonucleic acid sectionalisation tonus and establish designate step which involves nucleophilic attacks, generating single- filum nicks in Mu desoxyribonucleic acid and joining it to tush deoxyribonucleic acid via one-step transesterification utensil. The latter part will concentrate o n the different characteristics in for individually one reversal weapon in replicative alternate, the curiosity product is duplication of jumping gene assume in twain(prenominal) rear end and army deoxyribonucleic acid while in conservative surrogate, a simple insertion of jumping gene is created in the come out deoxyribonucleic acid.1. Characteristics of phage MuPhage, derived from the Greek word phagein, literally means to eat. Bacteriophage Mu was named as such(find out who did) due its disposition of infecting and inducing high levels of mutation in entertain bacteria Escherichia coli., hence the name Mu for mutator. The dual nature of Mu jumping gene and virus has do it as the archetypal simulate of studying phage genetics. Bacteriophage Mu is a temperate phage of E. coli which employs the about-face mechanism in its life cycle. Transposition can either be conservative (excising the jumping gene and inserting it into bacterial chromosome) or replicati ve (transposon copies be produced in twain transposon and bacterial chromosome). Both mechanisms will be discussed extensively later in this article. irrelevant the phage , insertion of Mu genome into the score invest proceeds in a randomly manner which makes it an excellent mutator.Fig. 1 The life cycle of bacteriophage Mu(5).The life cycle of phage Mu is shown schematically in Fig. 1 above. Bacteriophage Mu infect susceptible host cell by adsorption and hence, injects its analogue viral genome. Once inside the host cell, the linear genome does non circularized(4,5,19), impertinent in phage . In either case of lytic or lysogenic phase, Mu integrates its deoxyribonucleic acid into the host genome via conservative setback(16,19). This is go ond differently in phage where the infecting phage desoxyribonucleic acid will be integrated into host genome scarce during lysogenization(19). An enzyme called transposase, encoded by MuA gene in the phage genome, is absolutely cruci al to tolerate out this conservative transposition step. Phage DNA is inserted at septuple sites in a bacterial genome which lead to the assumption that the insertion draw by a random manner(8). However, on that transfer argon several factors that decide ass site selection such as MuA protein efficiency and transposition immunity(15).After integration, Mu commonly adopts a quiescent prophage lifestyle(lysogenic phase). The preference between lysogenic and lytic phase in Mu life cycle is dep kiboshent on its inactiveness in the lysogen and lysogenic repressers. However, lysogens of Mu phage sometimes enter the lytic phase though this is a r ar event. When induced, usually by using temperature-sensitive repressor mutants of phage Mu and subject it at 42C, the lysogen will enter lytic cycle. When the lysogenic repressor is inactivated, Mu transposes via replicative transposition, producing copies of phage genome which will be packaged into new virions. The virions then lyse the host cell and infect new hosts. Bacteriophage Mu virions comprised of icosahedral head(diameter 54nm), a baseplate, a contractile tail and six short tail fibres(5).Fig. 2 simplify cartoon illustrating furtherance of Mu genome. Typical length of phage Mu DNA is some 37kb long. Additional 2 kb of host DNA is incorporated during DNA packaging which is shown as flanking each bar of the integrated Mu genome, with most of it at the business end. Unique sequences of host DNA and at the right end of the packaged DNA is dependent on initiation site of packaging in the host DNA(24).Fig. 3 Physical and genetic map of bacteriophage Mu. Solid black lines represent Mu DNA while the boxes at the dickens ends usher flanking host DNA sequences. Mu genes (indicated in block letters) and their stoping translational products ar as indicated(19).A typical size of wild-type phage Mu DNA is about 37.5 kb, however each phage capsid can lenify up to 39 kb long. Phage genome has a pac site which serves as the starting point in packaging of the phage DNA, turn up within attL(5). The initiation partition by phage enzyme terminase pop offs upstream of the phage pac site, which includes host sequence of about 50-150bp flanking the left end. Second division initiated when a complete filling of capsid is achieved, which includes 0.5 kb to 2 kb of host sequence flanking the right end(1). Genetic and physical map of phage Mu is illustrated in Fig. 3. Bacteriophage Mu utilizes headful mechanism strategy, which confer variable lengths of host DNA flanking the left ends of Mu DNA depending on the initiation site of genome packaging(Fig. 2).2. Transposition mechanism(E)(D)(C)(B)(A)Fig. 4 Modes of bacteriophage Mu transposition. (A), (B) and (C) argon the common steps in some(prenominal) conservative and replicative transposition of phage Mu. In conservative and replicative transposition, phage Mu will follow-up step (D) and (E) respectively. Curved arrows indicate nucleophile at tack, takering the 3-OH ends to the staggered 5-phosphate ends of target DNA. Dentate lines (XXXX) indicate target DNA sequences which ar duplicated during transposition (16).Numerous in vitro studies digest been conducted to study the mechanism of transposition, and usually mini-Mu components are used. A minimal Mu segment consists of a selectable gene, a plasmid DNA reproduction origin and essential Mu ends(2). The mechanism of transposition is discussed in respect to an in vitro system from this point onward unless stated differentwise. Following discussion on transposition mechanism are based on Shapiro ideal(22) as it has been widely accepted as the well-heeled pattern in this field.The current known modes of transposition is divided into both non-replicative (conservative) and replicative transposition. Both strategies utilize the same mechanism up to point (Fig. 4C) where each strategy employs different mechanism, producing different end products. A simple inse rtion of transposon is generated in target DNA by conservative transposition (Fig. 4D) while two copies of transposon formed in twain presenter and target DNA by replicative transposition (Fig. 4E). Point A to C are considered as the similar features in both conservative and replicative transposition while point D and E is the distinction between the two modes of transposition. Therefore, mechanisms involved in point A,B and C are discussed in context of both replicative and conservative transposition, which comprises of DNA cleavage step and strand ship step. Sequential stages of both cleavage and strand transfer steps are illustrated in Fig. 4.2.1 Donor DNA cleavage step dickens decisive chemical steps in both transposition pathways are bestower DNA cleavage step and DNA strand transfer step(5,8). The conferrer DNA cleavage step is initiated when water molecules within an active site act as nucleophiles, and attack phosphodiester bond in DNA ground tackle at each of the t ransposon end(4,5). The cleavage step involves a invest hydrolysis of phosphodiester bond by water, and not by covalent enzyme-DNA fair(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 end of the cleavage step. Strand transfer outlets in fusion of target and bestower DNA, which forms an intermediate molecule (8). The process (simplified in Fig. 4C) follows the Shapiro model(22).Bacteriophage-encoded proteins, MuA protein (transposase) and MuB protein (ATPase) are required for transposition. Other requirements to procure efficiency of transposition are accessory proteins such as host-encoded DNA bending proteins called hydroxyurea (HU) and integration host factor (IHF)(8). The inverted repeats at the end of donor DNA, and target sequence on bacterial chromosome are overly important in transposition mechanism. The assembly of higher order protein-DNA interwovenes called transposome has be en determine by in vitro studies(6).A three-site synaptic involved called the LER obscure comprising right and left ends of Mu and transpositional enhancer, was formed in the beginning of transposition in vitro(23). MuA protein binds to MuA salad dressing site at the ends of Mu DNA as monomer, and subsequently function as tetramer of MuA (transposase). Host IHF and HU protein were found to aid in organization and stabilisation of LER complex.The LER complex is relatively unstable and so, is rapidly converted into stable synaptic complex (SSC), also known as type 0 complex(17). This is the critical checkpoint in the beginning any chemical reply is carried out as it is the rate-limiting step of cleavage reaction(6). A stable synapse between tetramer of MuA and the two ends of Mu DNA is made only no cleavage is initiated yet at this point. Nonetheless, the active site is structurally occupied to the region around the scissile phosphate while the flanking DNA are destabilized upon organic law of the SSC complex(6). In addition to formation of a stable synapse, the Mu ends require to be properly-oriented, a super coiled DNA topology, and accessory DNA sites are also important to proceed to the next step. Formation of SSC usually is short-lived in mien of Mg2+ but can be accrued in presence of suitable divalent cations such as Ca2+,which promotes the formation of SSC(8,17).Next, SSC is converted into a type 1 transposome complex, also called as cleaved donor complex(CDC)(9). The 3 ends of Mu DNA are nicked in presence of Mg2+. Two subunits of MuA tetramer, that are associated with the sites that undergo cleavage, assemble in trans arrangement which favours the strand transfer reaction(5). The formation of CDC can then be thought as the result of donor DNA cleavage step. Type 1 transposome complex exhibits greater stability than the type 0 complex though MuA forms structural and available pith in both transposome complexes(6). In addition of stably bound t etramer of MuA proteins, there are loosely associated MuA proteins present in the CDC as well. In absence seizure of MuB protein, MuA tetramer is unable to promote strand transfer reaction unless these extra MuA proteins are present. MuB protein is an ATP-dependent DNA- dressing protein, which also acts as an allosteric activator of Mu transposase (MuA proteins)(21). Transposition can still proceed in absence of MuB proteins, but MuA protein by itself is only 1% efficient(3).2.2 Strand transfer stepA hallmark of this step is the formation of strand transfer complex (STC), also known as type 2 transposome complex. The end product of STC is formation of a pronged molecule(Shapiro intermediate) which is characterized by a covalent interaction between donor DNA and target DNA via 5bp single-stranded gaps and its structure(22). MuB protein first captures a target molecule and bring it to the vicinity of the transposome complex, forming a TC complex(6). Formation of TC complexes rapidly undergo one-step transesterification reaction, which is the rate-limiting step in the strand transfer step. Interestingly, recruiting of target molecules by MuB proteins and formation of TC complexes can occur at several time point during the reaction pathway(6). This is a particularly efficient step to maximize transposition say-so as it would speed up rate of strand transfers during transposition.The free 3-OH ends produced from the cleavage step act as nucleophile and attack phosphates of target DNA at the 5 ends. 5-nucleotides long offset nicks are made in the target DNA, generating a staggered arrangement(3). At this stage, the MuA proteins(transposase) are still tightly bound to the branched molecule with single stranded gaps. This pose an obstruction for the assembly of reappearance fork by host replication factors. The structure of the branched 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 transposition. A widely accepted model is that the resolving of this co-integrate molecule by a special resolvase complex leads to replicate copies of transposon existence made in both donor and target site(REFerence). This is by definition, a replicative transposition pathway. Thus, the strand transfer complex 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, producing cointegrates(13).This handing over from transposome complex to a replisome results in duplication of 5-bp target DNA sequences flanking both ends of Mu DNA. Alternatively, if the bacteriophage Mu is to enter the conservative pathway, the co-integrate molecule is repaired or processed without perform Mu DNA replication. The end product of STC in a conservative transposit ion is a simple insertion of single mini-Mu element inserted into the target DNA(8). However, the mechanism of this model is poorly understood.Fig. 5 Transposome complexes involved during DNA cleavage complex and DNA strand transfer. (A) A plasmid (gray line) bearing donor mini-Mu element (black line) DNA in the in vitro system is negatively coiled. (B) In presence of host HU protein, Mu A protein bind to the two ends of Mu DNA forming a stable synaptic complex (not shown). Assembly of MuA tetramer produces a nick at each ends of Mu DNA, creating a cleaved donor complex (CDC). (C) Nicked 3 ends of Mu DNA are conjugate together to target DNA in presence of MuB protein forming a strand transfer complex (STC). MuA tetramer is still tightly bound to the Mu ends in the STC. (D) In replicative transposition, a cointegrate molecule is produced when replication of target DNA initiated from the 3 Mu ends by host replication machinery (13).3. Replicative transpositionReplicative transpositio n was first suggested by Ljungquist and Bukhari (1977) to occur in situ after induction of lysogens, which means that the Mu prophage was not excised from host chromosome during transposition(14). The lysogens were digested with breastwork enzymes which cleaves both host and Mu DNA at specific restriction sites. Two of the fragments from the restriction digests contain both host and Mu DNA, which corresponds to junctions between host and prophage DNA, suggesting that prophage DNA is replicated in situ of host chromosome(19). Several genetic and biochemical predictions made in the Shapiro model pack been demonstrated in both in vivo and in vitro studies, hence this model is accepted as a plausible mechanism to explain transposition in phage Mu.Numerous techniques have been do to study the direction of replication of Mu DNA during transposition. Results obtained by annealing of Okazaki fragments to scattered strands of Mu DNA shows that more than 80% of Mu molecules replication pr oceed from left to right end(11,19). Electron microscopical observation of mini-Mu element shows that replicating molecules in vitro replicate from both ends in equal probability(11,19). Replication of Mu DNA is accepted to be predominantly unidirectional, that is from left towards the right end(20). Intramolecular replication pathway can result in inversion, deletion, and simple insertion while intermolecular events can produce co-integrate molecules(19). In the case of Mu transposition, formation of co-integrate molecule needs to be end in order to produce two replicons one molecule contains transposon and target DNA while another molecule contains transposon and donor DNA(10).4. Conservative transpositionThe main characteristic of conservative transposition is that phage DNA is not replicated prior to integration. Upon infection of a susceptible host cell (usually E. coli), Mu employs conservative, or also called non-replicative transposition to transfer its genome to the targe t site. As discussed earlier, conservative transposition pathway follows single strand nicks at the 3 ends of Mu DNA, of which the exposed 3-OH ends join to the staggered cut target DNA at the 5ends forming a co-integrate molecule. The co-integrate or so-called Shapiro intermediate is repaired and generates a simple insertion in the target DNA though the mechanism is still poorly understood.Shapiro model emphasized on single-stranded nicks at Mu ends, joining of Mu to a staggered double-strand issue in target DNA, formation of an intermediate molecule, and shedding of heterogeneous of front host DNA sequences after ligation in conservative pathway(22). On the other hand, Morisato and Kleckner (1984) proposed a different mechanism based on results with Tn10 transposition. Their model is double-stranded cleavages at the transposon ends generating an excised transposon, which then circularizes via ligation on one of the strands(18). It predicts shedding of host sequences from the Mu DNA ends before ligation into the new target DNA. Study of Mu transposition using plasmid substrates in vitro produced results in favour of the Shapiro model, and hence this model has been widely accepted and used in studies.Fig. 6 A model of conservative transposition which utilizes double-strand cleavages during integration. (A) Transposase bind to the inverted repeats at Mu-host boundary sites and cleaves off the transposon away. (B) Transposase made a staggered cut at target sequence of which exposed 3-OH ends of transposon attacks 5-phosphate ends of the host (not shown). The transposon then joins to the host sequence. Duplicated target sequence of 5-bp are completed by host replication machinery (7).The debate on single-strand or double-strand cleavage however does not end there. If phage Mu were to utilize the Shapiro model of transposition during integration (the well-established cointegrate mechanism), the flanking host sequences would remain bound to Mu ends. This would cl early pose a problem as subsequent target-primed replication of the linear integrant would not work, or simply break the chromosome(1). Evidently, results from in vitro experiments are against this as the transposition end products contain transposon, suggesting a complete transposition process have been accomplished. So, does the infecting Mu DNA utilize the Shapiro model where the cointegrate molecule gets processed and repaired, prior to replication 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 eventually means, there is no need for resolve by replication?An in vitro experiment was done by Au et al. (2006) to observe the fate of flanking host DNA sequences upon phage Mu infection. Specific markers specific to the infecting phage Mu DNA as well as the donor host (lacZ/proB) were used. These markers w ere 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 time point (approximately at import 8), which correspond to the integration time point of Mu. Subsequent expression of lacZ and proB were detected maximally at minute 15, significantly reduced at minute 30 and by minute 50, expression were halted(1). Maximal expression at minute 15 most likely corresponds to climax of integration of the infecting phage population. These findings powerfully suggest that flanking sequences get integrated together with Mu DNA into the new target site and are subsequently, removed by a special mechanism(which explained the insensible(p) expression at minute 50). This then proves that infecting phage Mu employs an alternate cointegrate mechanism (also called as nick-join-process mechanism) in conservative transposit ion pathway, where the Mu DNA undergo single-strand nicks, joins to the target DNA, and repaired before replication of the 5-bp gap left by the flanking sequence(1). The mechanism of remotion and repair of host flanking sequence however, remains ambiguous.ConclusionDual nature of bacteriophage Mu, a transposable element and a virus, is certainly raise but what is more fascinating is that it utilizes both replicative and non-replicative transposition throughout its life cycle. The former mechanism produces a transposon copy in both donor and target DNA while the latter usually generates a simple insertion of transposon in the target DNA, leaving a gap in the host DNA which most likely will get degraded.In the early stages, both replicative and conservative transposition pathway share a similar mechanism. Regardless of the transposition pathway, infecting Mu DNA during the first round of infection will integrate its DNA into the target chromosome via two critical steps donor DNA clea vage step and strand transfer step. Mu uses a phosphoryl transfer involving nucleophilic attacks of water on phosphodiester bonds of Mu DNA, producing single-strand nicks. A gage nucleophilic attack by exposed 3-ends of Mu DNA on 5-ends of target phosphodiester bonds, which then joins the Mu DNA to target DNA via one-step transesterification mechanism. A series of transposome complexes are formed throughout these processes including Mu-encoded MuA proteins(transposase) and MuB proteins(ATPase). A cointegrate is produced in both pathways but in replicative transposition, this intermediate molecule is resolved producing two replicons with transposon copy in each molecule. In conservative transposition, the cointegrate is repaired generating a simple insertion in the target DNA. Hence, it is more accurate to name conservative transposition as nick-join-process rather than the conventional cut-and-paste mechanism as the latter suggest double-strand nicks at the transposon end, which ha s been proven inaccurate by in vitro experiments. Both transposition pathways have been compared extensively in this review but much of functional core of the mechanisms remain to be understood.(2944 words)