10. Coordinating synthesis of the lagging and leading strands

13.10 Coordinating synthesis of the lagging and leading strands




Figure 13.17 Leading and lagging strand polymerases move apart.
Animated figure

A major problem of the semidiscontinuous mode of replication is illustrated in Figure 13.17: how is synthesis of the lagging strand coordinated with synthesis of the leading strand? The new DNA strands are growing in opposite directions. One enzyme unit is moving with the unwinding point and synthesizing the leading strand. The other unit is moving "backwards," relative to the DNA, along the exposed single strand. When synthesis of one Okazaki fragment is completed, synthesis of the next Okazaki fragment is required to start at a new location approximately in the vicinity of the growing point for the leading strand. This requires a translocation relative to the DNA of the enzyme that is synthesizing the lagging strand.


As the replisome moves along DNA, unwinding the parental strands, it elongates the leading strand. Periodically the primosome activity initiates an Okazaki fragment on the lagging strand. We can propose two types of model for what happens to the DNA replicase when it completes synthesis of an Okazaki fragment. It might dissociate from the template, so that a new complex must be assembled to elongate the next Okazaki fragment. Or the same complex may be reutilized.


We can now relate the subunit structure of DNA polymerase III to the activities required for DNA synthesis and propose a model for its action. The holoenzyme is a complex of 900 kD that contains several components:


  • a catalytic core, including the α subunit;
  • a dimerization component (τ) that links two cores;
  • a processivity component (β) that keeps the polymerase on the DNA;
  • and a clamp loader (γ) that places the processivity subunits on DNA.



Figure 13.18 DNA polymerase III holoenzyme assembles in stages, generating an enzyme complex that synthesizes the DNA of both new strands.

A speculative model for the assembly of DNA polymerase III is depicted in Figure 13.18. The holoenzyme assembles on DNA in three stages:



  • A β dimer plus a γ complex recognizes the primer-template to form a preinitiation complex. In this reaction, the γ complex cleaves ATP and transfers β subunits to the primed template. The pair of β subunits forms a "clamp" that binds around the DNA and ensures processivity. The γ complex is a "clamp loader" that uses hydrolysis of ATP to drive the binding of β to DNA.
  • Binding to DNA changes the conformation of the site on β that binds to the γ complex, and as a result it now has a high affinity for the core polymerase. This enables core polymerase to bind, and this is the means by which the core polymerase is brought to DNA. The "core enzyme" contains subunits α , ε , and θ . The α subunit has a basic ability to synthesize DNA, the ε subunit has the 3′ V5′ proofreading exonuclease, and θ may be required for assembly. (The processivity of the core by itself is low; but the β clamp ensures that it functions processively on the DNA.)
  • A τ dimer binds to the core polymerase, and provides a dimerization function that binds a second core polymerase (associated with another β clamp). The holoenzyme is asymmetric, because it has only 1 γ complex. This γ complex is responsible for adding a pair of β dimers to each parental strand of DNA.

Each of the core complexes of the holoenzyme synthesizes one of the new strands of DNA. Because the clamp loader is needed for unloading the β complex from DNA, the two cores have different abilities to dissociate from DNA. This corresponds to the need to synthesize a continuous leading strand (where polymerase remains associated with the template) and a discontinuous lagging strand (where polymerase repetitively dissociates and reassociates). The γ complex is associated with the core polymerase that synthesizes the lagging strand, and plays a key role in the ability to synthesize individual Okazaki fragments.




Figure 13.19 The b subunit of DNA polymerase III holoenzyme consists of a head to tail dimer (the two subunits are shown in red and orange) that forms a ring completely surrounding a DNA duplex (shown in the center). Photograph kindly provided by John Kuriyan.
Multiple figure

The β dimer makes the holoenzyme highly processive. β is strongly bound to DNA, but can slide along a duplex molecule. The crystal structure of β shows that it forms a ring-shaped dimer. The model in Figure 13.19 shows the β-ring in relationship to a DNA double helix. The dimer surrounds the duplex, providing the "sliding clamp" that allows the holoenzyme to slide along DNA. The structure explains the high processivity Xthere is no way for the enzyme to fall off! Because the clamp is a circle of subunits surrounding DNA, its assembly or removal requires the use of an energy-dependent process by the clamp loader.




Figure 13.20 Each catalytic core of Pol III synthesizes a daughter strand. DnaB is responsible for forward movement at the replication fork.

The asymmetric dimer model for DNA polymerase III suggests the structure for the replication fork that is illustrated in Figure 13.20. A catalytic core is associated with each template strand of DNA. The holoenzyme moves continuously along the template for the leading strand; the template for the lagging strand is "pulled through," creating a loop in the DNA. DnaB creates the unwinding point, and translocates along the DNA in the "forward" direction (moving 5′ V3′ on the template for the lagging strand).


DnaB contacts the τ subunit(s) of DNA polymerase. This establishes a direct connection between the helicase-primase complex and the polymerase itself. This link has two effects. One is to increase the speed of DNA synthesis by increasing the rate of movement by DNA polymerase core by 10 . The second is to prevent the leading strand polymerase from falling off, that is, to increase its processivity.


Synthesis of the leading strand creates a loop of single-stranded DNA that provides the template for lagging strand synthesis, and this loop becomes larger as the unwinding point advances. After initiation of an Okazaki fragment, the lagging strand core complex pulls the single-stranded template through the β clamp while synthesizing the new strand. The single-stranded template must extend for the length of at least one Okazaki fragment before the lagging polymerase completes one fragment and is ready to begin the next.




Figure 13.21 Core polymerase and the b clamp dissociate at completion of Okazaki fragment synthesis and reassociate at the beginning.

What happens to the loop when the Okazaki fragment is completed? When a Pol III holoenzyme meets a nick in DNA, the core complex and clamp dissociate from the β sliding clamp. The core can then reassociate with a new β subunit elsewhere. Figure 13.21 suggests that this represents the reaction that occurs at the end of an Okazaki fragment. The core complex will dissociate when it completes synthesis of each fragment, releasing the loop. The core complex then associates with a β clamp to initiate the next Okazaki fragment. Probably a new β clamp will already be present at the next initiation site, and the β clamp that has lost its core complex will dissociate from the template (with the assistance of the γ complex) to be used again. So the lagging strand polymerase will probably transfer from one β clamp to the next in each cycle, without dissociating from the replicating complex.


What is responsible for recognizing the sites for initiating synthesis of Okazaki fragments? In oriC replicons, the connection between priming and the replication fork is provided by the dual properties of DnaB: it is the helicase that propels the replication fork; and it interacts with the DnaG primase at an appropriate site. Following primer synthesis, the primase is released. The length of the priming RNA is limited to 8 V14 bases. Apparently DNA polymerase III is responsible for displacing the primase.




Figure 13.22 Synthesis of Okazaki fragments requires priming, extension, removal of RNA, gap filling, and nick ligation.

We can now expand our consideration of the actions involved in joining Okazaki fragments, as illustrated in Figure 13.22. The complete order of events is uncertain, but must involve synthesis of RNA primer, its extension with DNA, removal of the RNA primer, its replacement by a stretch of DNA, and the covalent linking of adjacent Okazaki fragments.


The figure suggests that synthesis of an Okazaki fragment terminates just before the start of the RNA primer of the preceding fragment. When the primer is removed, there will be a gap. The gap is filled by DNA polymerase I; polA mutants fail to join their Okazaki fragments properly. The 5′ V3′ exonuclease activity removes the RNA primer while simultaneously replacing it with a DNA sequence extended from the 3′ VOH end of the next Okazaki fragment. This is equivalent to nick translation, except that the new DNA replaces a stretch of RNA rather than a segment of DNA. In mammalian systems (where the DNA polymerase does not have a 5′ V3′ exonuclease activity), Okazaki fragments are removed by a two-step process. First RNAase HI (an enzyme that is specific for a DNA-RNA hybrid substrate) makes an endonucleolytic cleavage; then a 5′ V3′ exonuclease called FEN1 removes the RNA.


Once the RNA has been removed and replaced, the adjacent Okazaki fragments must be linked together. The 3′ VOH end of one fragment is adjacent to the 5′ Vphosphate end of the previous fragment. The responsibility for sealing this nick lies with the enzyme DNA ligase. Ligases are present in both prokaryotes and eukaryotes. Unconnected fragments persist in lig V mutants, because they fail to join Okazaki fragments together.




Figure 13.23 DNA ligase seals nicks between adjacent nucleotides by employing an enzyme-AMP intermediate.

The E. coli and T4 ligases share the property of sealing nicks that have 3′ VOH and 5′ Vphosphate termini, as illustrated in Figure 13.23. Both enzymes undertake a two-step reaction, involving an enzyme-AMP complex. (The E. coli and T4 enzymes use different cofactors. The E. coli enzyme uses NAD [nicotinamide adenine dinucleotide] as a cofactor, while the T4 enzyme uses ATP.) The AMP of the enzyme complex becomes attached to the 5′ Vphosphate of the nick; and then a phosphodiester bond is formed with the 3′ VOH terminus of the nick, releasing the enzyme and the AMP.


The system that synthesizes eukaryotic DNA in animal cells has two DNA polymerases. DNA polymerase α exists as a complex consisting of a 180 kD catalytic subunit, associated with other subunits, including two smaller proteins that provide a primase activity. DNA polymerase δ is less well characterized. The counterparts of these enzymes in yeast, DNA polymerases I and III, are coded by essential genes.


Initiation at the SV40 origin requires a product of the virus, the T antigen, to bind to the origin and initiate the process of strand separation. Replication factor A is a single-strand binding protein that allows T antigen to unwind the SV40 DNA more extensively. The DNA polymerase α /primer complex initiates the synthesis of both DNA strands. The priming reaction is unusual: it starts with RNA, like other priming reactions, but the RNA primer is extended by the DNA polymerase activity to give a short (3 V4 base) DNA sequence, called iDNA. Replication factor C binds to the 3′ end of the iDNA and loads DNA polymerase δ and PCNA. (PCNA is called proliferating cell nuclear antigen for historical reasons. It acts as a processivity factor that tethers DNA polymerase δ to the template.) In this system, SV40 T antigen initiates DNA unwinding, functions as a helicase, and loads the replication apparatus; separate proteins fulfill these roles in the eukaryotic host cell.


At one time, it was thought that DNA polymerase α synthesized the lagging strand, while DNA polymerase δ synthesized the leading strand, but now it seems that the role of DNA polymerase α is to initiate, and the role of DNA polymerase δ is to elongate, both strands. So the same basic sequence of events occurs during initiation of both the leading strand and the Okazaki fragments of the lagging strand. We do not know whether the same individual complex of DNA polymerase α /primase that initiates the leading strand is used for the lagging strand, but we have a model for the function of a eukaryotic replisome as it proceeds along the template.


This model suggests that a replication fork contains 1 complex of DNA polymerase α /primase and has 2 complexes of DNA polymerase δ . The two complexes of DNA polymerase δ behave in the same way as the two complexes of DNA polymerase III in the E. coli replisome: one synthesizes the leading strand, and the other synthesizes Okazaki fragments on the lagging strand. The processivity of DNA polymerase δ is maintained by PCNA. The crystal structure of PCNA closely resembles the E. coli β subunit: a trimer forms a ring that surrounds the DNA. Although the sequence and subunit organization are different from the dimeric β clamp, the function is likely to be identical. The DNA polymerase δ of the lagging strand fills in the gap between Okazaki fragments after the exonuclease MF1 has removed the RNA. The enzyme DNA ligase I is specifically required to seal the nicks between the completed Okazaki fragments.




Genes VII
Genes VII
ISBN: B000R0CSVM
EAN: N/A
Year: 2005
Pages: 382

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