Replication strategies of bacteriophage
Studies of DNA replication in bacteriophage have been very valuable because of the insights that have been obtained into replication strategies, mechanisms and enzymology. However, the faithful and accurate replication of a genome is easily accomplished only if the genome is circular and is made of double-stranded DNA. If the genome is linear or if it is single-stranded or if it is made of RNA then special strategies are called for.
LINEAR GENOMES
If the genome is linear then it will progressively shorten with each round of replication unless the organism adopts a strategy for dealing with this problem.
The fact that DNA polymerase requires an RNA primer coupled with the fact that DNA polymerases are capable of synthesizing DNA only in the 5' -> 3' direction poses a critical problem for the replication of linear DNA molecules. Simply put - it is impossible to synthesize two exact copies of a parental molecule under these enzymological constraints. The problem boils down to one of how do you fill in the ends?
Consider the following:
Assume that replication of the linear molecule is initiated by the synthesis of RNA primers (red arrows) at each end. In the above example, these serve as primers for leading strand synthesis which copies each of the two parental strands. The end results are two daughter molecules each with an RNA-DNA hybrid polynucleotide chain (line 3).
However, when these RNA primers are removed, we are left with two molecules with single-stranded ends. These ends cannot be repaired or copied by DNA polymerase because of their polarity. Remember that no known DNA polymerase works in a 3' -> 5' direction.
If we now try and follow another round of replication using one of the original two daughters as the new parent, we see the full scope of the problem with replicating linear genomes:
After another round of replication, we recover one molecule that is identical to the parent but the other is not - it is shorter and has lost some genetic material.
So the problem with linear genomes is that they will progressively shorten with each round of replication unless some strategy is adopted to prevent this from occuring.
Two different strategies are described below for overcoming this problem: bacteriophage lambda circularises its chromosome; bacteriophage T7 forms concatemers.
OTHER GENOMES
If the genome is either a ssDNA or an RNA genome then the organism must use special enymes or strategies or some combination of the two in order to replicate. An example is discussed below in the replication of bacteriophage M13 which has ssDNA genome.
Rolling Circle Replication
Replication via theta forms is not the only method by which circular molecules can replicate their genetic information. Another method is rolling circle replication, though it generates linear copies of a genome rather than circular copies.
Consider a circular molecule of double-stranded DNA with a nick in one of the two phosphodiester backbones. As long as there is a free 3' OH end, this can serve as a template for DNA polymerase. When the 3' OH end is extended, the 5' end can be displaced in a manner analogous to the strand displacement reaction. Synthesis on this strand is also analogous to leading strand synthesis. The displaced strand can, in turn, serve as an template for replication as long as a suitable primer is available. Synthesis on this strand is analogous to lagging strand synthesis.
If synthesis continues in this manner, the consequence of this mechanism of replication can be the production of concatemer copies of the circular molecule. As a result, multiple copies of a genome are produced.
A rolling circle mode of replication is seen both during replication of bacteriophage lambda where rapid production of many copies of the genome is desired, and in the replication of bacteriophage M13 where only a single copy is produced each time.
View an Animation of Rolling Circle DNA Replication from a Spring 2000 Web Page Term Paper by Tara Cheung, Rebecca Frederick, Kristina Lamothe, Alicia Lee, and Kelly Watson at Carnegie Mellon University
Replication of Bacteriophage Lambda
Bacteriophage lambda contains a linear dsDNA genome. However, the ends of the genomic DNA are single-stranded and are cohesive, i.e. they are complementary to one another. The two cohesive ends - known as cos sites - are 12 nt in length.
After adsorption of the phage to the bacterial cell surface and injection into the cell, the chromosome circularizes by means of these complementary cohesive ends. This helps to protect it from degradation by bacterial exonucleases. Circularization is also an essential step if bacteriophage lambda chooses a lysogenic mode of growth.
Bacteriophage lambda replicates in two stages.
The gene O product has a function analogous to that of DnaA. It binds to repeated sequences at the origin and initiates melting of the two strands nearby. The gene P product has a function analogous to that of DnaC. It helps DnaB to bind to the "melted" DNA. Thereafter, the other components of a bacterial replisome can bind and replication ensues.
This mode of replication continues for 5 - 15 minutes after replication.
As concatemers are synthesized, they must be processed into linear molecules. This occurs by the action of Terminase which consists of two protein subunits coded by the lambda A and Nu1 genes. Gene A codes for a 74 kDa protein; Nu1 codes for a 21 kDa protein. Terminase recognizes the cos sites (in its double-stranded form) and cleaves them to generate new cohesive ends.
In vivo, processing of the concatemers also requires some of the other capsid proteins and there are length constraints on the amount of DNA that can be packaged. After the first cos site has been recognized, the second one must be located within 75% to 105% of the unit length of the phage chromosome.
The ability of the capsid to measure the amount of DNA that is packaged as well as recognizing specific sites is an important factor in the use of bacteriophage lambda as a cloning vector. Bacteriophage lambda derived cloning vectors can only be used to clone DNA fragments that are less than 15 kb in size (the actual size depends on the specific vector).
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Replication of Bacteriophage M13
Bacteriophage M13 (and other filamentous phage like it) has a circular ssDNA molecule in the capsid. When the phage attaches to an E. coli cell, this molecule is injected into the cell where most of it is coated with single-strand binding protein (SSB). Since bacteriophageM13 does not code for its own DNA polymerase, it must use the host cell machinery in order to replicate. It is, therefore, constrained by the requirements of the host cell replication machinery. Although its ssDNA genome is a perfect template for DNA synthesis, it is not such a suitable template either for RNA synthesis by either RNA polymerase or by primase.
[Snyder-Champness 7-12] [MVH24-36]
However, although most of the genome is single-stranded, one part of it forms a double-stranded hairpin. This region somehow can serve as a promoter for the host cell RNA polymerase, which transcribes a short RNA primer. Transcription also disrupts the hairpin. DNA PolIII can then take over and synthesizes a dsDNA molecule.
This dsDNA molecule is known as RFI - replicative form I.
[Box24-1a]
Further replication of RFI does not proceed by means of theta intermediates but by a type of rolling circle replication. The gp2 endonuclease, which is encoded by the phage gene 2, nicks the RFI DNA at a specific site (+ strand origin). Rolling circle replication now occurs with displacement of a single strand. Concatemers are not formed; rather the gp2 endonuclease cleaves a second time after one complete copy has been synthesized. Thus the products of this one round of replication are a ssDNA circular molecule (the displaced strand - ligated into a circle) and a dsDNA RFI molecule. The circular ssDNA molecule can now be duplicated by repeating this entire sequence.
[Box24-1b]
In order to synthesize the ssDNA strands that are to be packaged into the capsid, the displaced ssDNA molecules must be coated with a single strand binding protein - gp5 - which is coded by the phage gene 5. These molecules are then packaged into new phage capsids.
The fact that the life-cycle of filamentous phage such as M13 includes both a ssDNA phase and a dsDNA phase has been very useful for molecular biologists. Cloning vectors based on M13 have been developed which allow one to clone small DNA fragments and propagate them as phage particles. The dsDNA form permits routine cloning operations. The ssDNA form is ideally suited for the Sanger sequencing protocol and for many protocols for site-directed mutagenesis.
Replication of Bacteriophage T7
Bacteriophage T7 has a linear dsDNA genome, 39,937 bp in size. Replication initiates at a site located approx. 5900 bp from the left end of the phage and proceeds bidirectionally.
| Although this specific case is different from the one drawn above, the problem is exactly the same. You should draw a diagram of the replication of T7 using pencil and paper. You will see that a bidirectional model of replication will not result in two complete daughter molecules in this case either. |
The solution to the problem of replicating T7 lies in the left and right ends of the genome. The first 160 bp at the left end are identical with the final 160 bp at the right end. It is this terminal redundancy that is the key to its replication. The following cartoon shows the products of one round of replication. Although that the extent of the single-stranded region is identical to that of the terminal repeat in this picture, this does not need to be the case.
The ssDNA at the right end (3' end after synthesis) of one T7 chromosome is able to anneal with ssDNA at the left end (also a 3' end after synthesis) of another. The remaining gaps can then be filled by DNA polymerase and ligated by DNA ligase. The resulting dimeric molecule can be cleaved in two again - but this time generating two 5' overhangs on each daughter, which are now able to act as templates for normal 5' -> 3' synthesis:
T7 encodes its own DNA ligase (gene 1.3), SSB (gene 2.5) and DNA polymerase (gene 5).
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RESOURCE MATERIAL |
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| VOET, VOET & PRATT |
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| STRYER |
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| LEHNINGER |
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| TAMARIN |
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| WEB SITES |
The following web sites were used for some of the individual links that have been incorporated into the lecture notes onbacteriophage and viruses. There are a lot of sites dealing with viruses and virology. Browse through them and learn from them but don't overdo it. There's lots more to this course than viruses!
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