Genes VII

A common feature of all DNA polymerases is that they cannot initiate synthesis of a chain of DNA de novo. Figure 13.11 shows the features required for initiation. Synthesis of the new strand can only start from a pre-existing 3′ VOH end; and the template strand must be converted to a single-stranded condition.

The 3′ VOH end is called a primer. The primer can take various forms, some of which we have discussed already in 12 The replicon in the context of the replicon. Types of priming reaction are summarized in Figure 13.12:

Figure 12.34 Replication of ColE1 DNA is initiated by cleaving the primer RNA to generate a 3 F-OH end. The primer forms a persistent hybrid in the origin region.

• A sequence of RNA is synthesized on the template, so that the free 3′ VOH end of the RNA chain is extended by the DNA polymerase. This is commonly used in replication of cellular DNA, and by some viruses. We discussed the example of ColE1 replication previously (see Figure 12.34).
• A preformed RNA pairs with the template, allowing its 3′ VOH end to be used to prime DNA synthesis. This mechanism is used by retroviruses to prime reverse transcription of RNA (see 16 Retroviruses and retroposons).

  Figure 12.16 The rolling circle generates a multimeric single-stranded tail.Animated figure

  Figure 13.5 Nick translation replaces part of a pre-existing strand of duplex DNA with newly synthesized material.

• A primer terminus is generated within duplex DNA. The most common mechanism is the introduction of a nick, as used to initiate rolling circle replication (see Figure 12.16). In this case, the pre-existing strand is displaced by new synthesis. (Note the difference from nick translation shown in Figure 13.5, in which DNA polymerase I simultaneously synthesizes and degrades DNA from a nick.)

  Figure 12.15 Adenovirus terminal protein binds to the 5 F end of DNA and provides a C-OH end to prime synthesis of a new DNA strand.

• A protein primes the reaction directly by presenting a nucleotide to the DNA polymerase. This reaction is used by certain viruses, as discussed previously in Figure 12.15.

Priming activity is required to provide 3′ VOH ends to start off the DNA chains on both the leading and lagging strands. The leading strand requires only one such initiation event, which occurs at the origin. But there must be a series of initiation events on the lagging strand, since each Okazaki fragment requires its own start de novo. Each Okazaki fragment starts with a primer sequence of RNA, ~10 bases long, that provides the 3′ VOH end for extension by DNA polymerase.

A primase is required to catalyze the actual priming reaction. This is provided by a special RNA polymerase activity that is the product of the dnaG gene. The enzyme is a single polypeptide of 60 kD (much smaller than RNA polymerase). The primase is an RNA polymerase that is used only under specific circumstances, that is, to synthesize short stretches of RNA that are used as primers for DNA synthesis. DnaG primase associates transiently with the replication complex, and typically synthesizes an 11 V12 base primer. Primers start with the sequence pppAG, opposite the sequence 3′ VGTC V5′ in the template.

(Some systems use alternatives to the DnaG primase. In the examples of the two phages M13 and G4, which were used for early work on replication, an interesting difference emerged. G4 priming uses DnaG, but M13 priming uses bacterial RNA polymerase. These phages have another unusual feature, which is that the site of priming is indicated by a region of secondary structure.)

There are two types of priming reaction in E. coli.

• The oriC system, named for the bacterial origin, basically involves the association of the DnaG primase with the protein complex at the replication fork.
• The φX system, named for phage φX174, requires an initiation complex consisting of additional components, called the primosome (see below).

Sometimes replicons are referred to as being of the φX or oriC type.

The types of activities involved in the initiation reaction are summarized in Figure 13.13. Although other replicons in E. coli may have alternatives for some of these particular proteins, the same general types of activity are required in every case. A helicase is required to generate single strands, a single-strand binding protein is required to maintain the single-stranded state, and the primase synthesizes the RNA primer.

DnaB is the central component in both φX and oriC replicons. It provides the 5′ V3′ helicase activity that unwinds DNA. Energy for the reaction is provided by cleavage of ATP. Basically DnaB is the active component of the growing point. In oriC replicons, DnaB is initially loaded at the origin as part of a large complex (see 13.12 Creating the replication forks at an origin). It forms the growing point at which the DNA strands are separated as the replication fork advances. It is part of the DNA polymerase complex and interacts with the DnaG primase to initiate synthesis of each Okazaki fragment on the lagging strand.

4-18-2000