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FIGURE 8-12 Removal of UNA primers from newly synthesized DNA. The sequential function of RNAse H, 5' exonucleasc, DNA polymerase, and DNA ligase during the removal of RNA primers is illustrated. DNA present prior to RNA prime* removal is shown in gray, the RNA primer is shown in green, and the newly synthesized ONA that replaces the RNA primer is shown in red.

strand requires only a single RNA primer. In contrast, the discontinuous synthesis of the lagging strand means that new primers fire needed for each Okazaki fragment. Because a single replication fork can replicate millions of base pairs, synthesis of the lagging strand can require hundreds to thousands of Okazaki fragments and their associated RNA primers.

Unlike the RNA polymerases involved in mRNA, rRNA, and tRNA synthesis {see Chapter 12), primase does not require specifics DNA sequences to initiate synthesis of a new RNA primer. Instead, primase is activated only when it associates with other DNA replication proteins, such as DNA belicase. These proteins are considered in more detail below. Once activated, primase synthesizes a RNA primer using the most recently exposed lagging strand template, regardless of sequence.

RNA Primers Must Be Removed to Complete DNA Replication

To complete DNA replication, the RNA primers used for the initiation must be removed and replaced with DNA (Figure 8-12), Removal of the RNA primers can be thought of as a DNA repsiir event and this process shares many of the properties of excision DNA repair, a process covered in detail in Chapter 9.

To replace the RNA primers with DNA, an enzyme called RNAse H recognizes and removes most of each RNA primer. This enzyme specifically degrades RNA that is base-paired with DNA (hence, the "H" in its name, which stands for hybrid in RNA:DNA hybrid). RNAse H removes all of the RNA primer except the ribonucleotide directly linked to the DNA end. This is because RNAse H can only cleave bonds between two ribonucleotides. The final ribonucleotide is removed by an exonnclease thai degrades RNA or DNA from their 5' end.

Removal of the RNA primer leaves a gap in the double-stranded DNA that is an ideal substrate for DNA polymerase—a primer:template junction (see Figure 8-12). DNA polymerase fills this gap until every nucleotide is base-paired, leaving a DNA molecule that is complete except for a break in the backbone between the 3'OH and 5' phosphate of the repaired strand. This "nick" in the DNA can be repaired by an enzyme called DNA ligase. DNA ligase uses a high-energy co-factor (such as ATP) to create a phosphodiester bond between an adjacent 5' phosphate and 3'OH. Only after all RNA primers are replaced and the associated nicks are sealed is DNA synthesis complete.

DNA Helicases Unwind the Double Helix in Advance of the Replication Fork

DNA polymerases are generally poor at separating the two Irase-paired strands of duplex DNA. Therefore, at the replication fork, a second class of enzymes, called DNA helicases, catalyze the separation of the two strands of duplex DNA. These enzymes bind to and move directionally along ssDNA using the energy of nucleoside triphosphate (usually ATP) hydrolysis to displace any DNA strand that is annealed to the bound ssDNA. Typically, DNA helicases that act at replication forks are hexa-meric proteins that assume the shape of a ring (Figure 8-13). These ring-shaped protein complexes encircle one of the two single strands at the replication fork near the single-stranded:double-stnmded junction.

figure 8-13 DNAhelkasesseparate the two strands of the double helix. When ATP is added to a DNA helicase bound io ssD^JA, the helicase moves with a defined polarity on the ssDNA In the instance illustrated, the DMA helicase has a 5'—»3/ polarity, This polarity means thai the DMA helicase would be bound to the lagging strand template at the replication fork.

Like DMA polymerases, DMA helicases act processively. Each time they associate with substrate, they unwind multiple base pairs oi DNA. The ring-shaped hexameric DNA helicases found at replication forks exhibit high processivity because they encircle the DNA. Release of the helicase from its DNA substrate therefore requires the opening of tho hexameric protein ring, which is a rare event. Alternatively, the helicase can dissociate when it reaches the end of the DNA strand that it has encircled.

Of course, this arrangement of enzyme and DNA poses problems for the binding of the DNA helicase to the DNA substrate in the first place. Thus, there are specialized mechanisms that assemble DNA helicases around the DNA in cells (see "Initiation of Replication" below). This topological linkage between proteins involved in DNA replication and their DNA substrates is a common mechanism to increase processivity.

Each DNA helicase moves along ssDNA in a defined direction. This property is a characteristic of each DNA helicase called its polarity (see Box 8-1, Determining the Polarity of a DNA Helicase). DNA helicases can have a polarity of either 5'—»3' or 3'—»5'. This direction is always defined according to Ihe strand of DNA bound (or encircled for a ring-shaped helicase) rather than the strand that is displaced. In the case of a DNA helicase that functions on the lagging strand template of the replication fork, the polarity is 5'—»3' to allow the DNA hnlicfise to proceed toward the duplex region of the replication fork (see Figure 8-13). As is true for all enzymes thai move along DNA in a directional manner, movement of the helicase along ssDNA requires the input of chemical energy. For helicases, this energy is provided by ATP hydrolysis.

Single-Stranded Binding Proteins Stabilize Single-Stranded DNA Prior to Replication

After the DNA helicase has passed, the newly generated single-stranded DNA must remain free of base-pairing until it can be used as a template for DNA synthesis. To stabilize the separated strands, single-stranded DNA binding proteins (designated SSBs) rapidly bind io the separated strands. Binding of one SSB promotes the binding of another SSB to the immediately adjacent ssDNA (Figure 8-14). This is called cooperative binding and occurs because SSB molecules bound to immediately adjacent regions of ssDNA can also bind to each other. This strongly stabilizes the interaction of the SSB with ssDNA making sites already occupied by one or more SSB molecules preferred over other sites.

Cooperative binding ensures that ssDNA is rapidly coated by SSB as it emerges from the DNA helicase. (Cooperative binding is a prop-

Box 8-1 Determining the Polarity of a DNA Helicase

The activity of a DNA helicase can be detected by its ability to displace one strand of a DNA duplex from another. In a typical DNA helicase assay, the substrate is composed of one short, labeled ssDNA annealed to one long, unlabeled ssDNA (typically the label ts radioactive 32P incorporated into the short ssDNA). Consider a large circular ssDNA (for example, 5,000 bases) hybridtzed to a short (200 bases), labeled linear ssDNA molecule (Box 8-1 Figure I). A DNA helicase will displace the short linear ssDNA from the large ssDNA circle. Separation of the strands can be detected by a change in electrophoretic mobility of the short, labeled ssDNA, in a nondenaturing agarose gel (see Chapter 20). After the gel is exposed to X-ray film to detect only the radiolabeled DNA, the position in the gel that the short DNA occupies can be determined. Wheri it ¡s hybridized to the ssDNA circle, the short ssDNA will co-migrate with the large ssDNA circle. In contrast, once the short ssDNA has been displaced from the ssDNA circle by DNA helicase, it will migrate according to its actual size, 200 bases.

A modification of this simple experiment can be used to determine the polarity oi a DNA helicase. Suppose there is a restriction enzyme cleavage site located asymmetrically within the base-paired region (Box 8-1 Figure 2). When this site is deaved it will generate a largely single-stranded, linear DNA with two regions of dsDNA of different lengths at each end. Remember that DNA helicases bind to ssDNA, not dsDNA. Thus, the only place that a DNA helicase can bind this new linear substrate is between the two dsDNA regions. Because of the polarity of DNA helicases, any given DNA helicase can displace only one of the two short ssDNAs. Because the two short ssDNA regions are of different lengths, the size of the released fragment will reveal which direction the DNA helicase moved along the ssDNA region of the linear substrate.

200 bases /radiolabeled) DMA

200 bases /radiolabeled) DMA

5,000 bases (unlabeled ssDNA circle)

O ® O ® C DKAtelicass x-ray film exposed to agarose gel o

BOX B-1 FIGURE t A biochemical assay for DMA helicase activity, (a) DNA substrate to detect helicase activity A 5,000 bp unlabeled ssDNA circular DNA is annealed to a 200-base radiolabeled DMA. For convenience the two molecules are not drawn to scale <b) To detect DNA helicase activity, the DMA substrate- is exposed to the DMA helicase (in this case with and without ATP). After the reaction, the resulting DNA molecules are Separated by agarose gel electrophoresis (nor denaturing). When the short, radiolabeled DNA is base-paired with the large ssDNA circle, both molecules will co-migrate as a large molecule. In contrast, after the DNA helicase has acted, the short radiolabeled ssDNA will migrate at a position consistent with the length of the short radiolabeled ssDNA. After exposure of the agarose gel to X-ray film, only tfie position of the radiolabeled DNA will be visible. As a control, the two DNA mctecules can be separated by boiling, which also causes denatuition of the base-paired region

Box 8-1 (Continued)

BOX 8-1 FIGURE 2 Abiochemical assay for DNA helicase polarity, (a) The

DMA substrate. The same DNA substrate illustrated in Figure 1 is cleaved with a restriction enzyme that leaves blunt ends The restriction enzyme is chosen to cleave asymmetrically, leaving 125-base and 75-base radiolabeled ssDNA fragments annealed to the ends of a 5,000-base unlabeled ssDNA. The 5' and 3' ends of the resulting DNA molecules are indicated, (b) An illustration of en X-ray film exposed to an agarose gel used to separate the DNA products after DNA helicase treatment ts shown. The substrate generated in part (a) can be incubated with a DNA helicase to determine its polarity. Results for a 5'-*3' and a 3'-»5' DNA helicase are shown. Boiling of the substrate indicates the consequences of complete denaturation of all base-pairing.

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