The Replication Fork

Both Strands of DNA Are Synthesized Together at the Replication Fork

Thus far we have discussed DNA synlhesis in a relatively artificial context. That is, at a primerrtemplate junction that is producing only one new strand of DNA. In the cell, both strands of the DNA duplex are replicated at the same time. This requires separation of the two strands of the double helix to create two template DNAs. The junction between the newly separated template strands and the unreplicated duplex DNA is known as the replication fork (Figure 8-11). The replication fork moves continuously toward the duplex region of unreplicated DNA, leaving in its wake two ssDNA templates that direct the formation of two daughter DNA duplexes.

The anti-parallel nature of DNA creates a complication for the simultaneous replication of the two exposed templates at the replication fork. Because DNA is only synthesized by elongating a 3r end, only one of the two exposed templates can be replicated continuously as the replication fork moves. On this template strand, the polymerase simply "chases" the replication fork. The newly synthesized DNA strand directed by this template is known as the leading strand.

Synthesis of the new DNA strand directed by the other ssDNA template is more problematic. This template directs the DNA polymerase lo move in the opposite direction of the replication fork. The new DNA strand directed by this template is known as the lagging strand, As shown in Figure 8*11, this strand of DNA must be synthesized in a discontinuous fashion.

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" pair fingers leading strand lagging strand direction of ieading strand Polymerase movement leading strand direction of ieading strand Polymerase movement

lagging strand direction of lagging strand polymerase movement direction of lagging strand polymerase movement overall direction ci DNA replication replicated DNA

unrepticated DNA

FIGURE 8-11 The replication fork. Newly synthesized DNA is indicated in red and RNA primers ate indicated in green The Okazaki fragments shown ere ertfictalfy short for illustrative purposes, lfi the cell, Okazaki fragments can vary between lOO to greater than t,000 bases.

Although the leading strand DNA polymerase can replicate its template as soon as it is exposed, synthesis of the lagging strand must wait for movement of the replication fork to expose a substantial length of template before it can he replicated. Each time a substantial length of new lagging strand template is exposed, DNA synthesis is initiated and continues until it reaches the 5' end of the previous newly synthesized stretch of lagging strand DNA.

The resulting short fragments of new DNA formed on the lagging strand are called Okazaki fragments and can vary in length from 1.00U to 2,000 nucleotides in bacteria and too to 400 nucleotides in eukary-otes. Shortly after being synthesized, Okazaki fragments are covalently joined together to generate a continuous, intact strand of new DNA. Okazaki fragments are, therefore, transient intermediates in DNA replication.

The Initiation of a New Strand of DNA Requires an RNA Primer

As described above, all DNA polymerases require a primer with a free 3JOH. They cannot initiate a new DNA strand de novo. How are new strands of DNA synthesis started? To accomplish this, the cell takes advantage of the ability of RNA polymerases to do what DNA polymerases cannot: start new RNA chains de novo. Primase is a specialized RNA polymerase dedicated to making short, RNA primers (5-10 nucleotides long) on an ssDNA template, These primers are subsequently extended by DNA polymerase. Although DNA polymerases incorporate only deoxyribonucleotides into DNA, they can initiate synthesis using either an RNA primer or a DNA primer annealed to Ihe DNA template.

Although both the leading and lagging strands require primase to initiate DNA synthesis, the frequency of primase function on the two strands is dramatically different (see Figure 8-11). Each leading



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