DNA gyrase

Moves ahead of the replication fork, making and resealing breaks in the double-helical DNA to release torque that builds up as a result of unwinding at the replication fork

DNA primase

Synthesizes short RNA primers to provide a 3'-OH group for attachment of DNA nucleotides

DNA polymerase III

Elongates a new nucleotide strand from the 3'-OH group provided by the primer

DNA polymerase I

Removes RNA primers and replaces them with DNA

DNA ligase

Joins Okazaki fragments by sealing nicks in the sugar-phosphate backbone of newly synthesized DNA

phosphodiester bond without adding another nucleotide to the strand (IFigure 12.14d). Some of the major enzymes and proteins required for replication are summarized in Table 12.4.

Concepts 9

After primers are removed and replaced, the nick in the sugar-phosphate linkage is sealed by DNA ligase. More information on helicase, primase, and single-strand-binding proteins

The replication fork Now that the major enzymatic components of elongation—DNA polymerases, helicase, primase, and ligase—have been introduced, let's consider how these components interact at the replication fork. Because the synthesis of both strands takes place simultaneously, two units of DNA polymerase III must be present at the replication fork, one for each strand. In one model of the replication process (I Figure 12.15), the two units of DNA polymerase III are connected, and the lagging-strand template loops around so that, as the DNA polymerase III complex moves along the helix, the two antiparallel strands can undergo 5': 3' replication simultaneously.

In summary, each active replication fork requires five basic components:

1. helicase to unwind the DNA,

2. single-strand-binding proteins to keep the nucleotide strands separate long enough to allow replication,

3. the topoisomerase gyrase to remove strain ahead of the replication fork,

4. primase to synthesize primers with a 3'-OH group at the beginning of each DNA fragment, and

5. DNA polymerase to synthesize the leading and lagging nucleotide strands. Additional information about the mechanism of replication and an animation of a replication fork

Termination In some DNA molecules, replication is terminated whenever two replication forks meet. In others, specific termination sequences block further replication. A termination protein, called Tus in E. coli, binds to these sequences. Tus blocks the movement of helicase, thus stalling the replication fork and preventing further DNA replication.

The fidelity of DNA replication Overall, replication results in an error rate of less than one mistake per billion nucleotides. How is this incredible accuracy achieved? No single process could produce this level of accuracy; a series of processes are required, each catching errors missed by the preceding ones (I Figure 12.16).

DNA polymerases are very particular in pairing nucleo-tides with their complements on the template strand. Errors in nucleotide selection by DNA polymerase arise only about once per 100,000 nucleotides. Most of the errors that do arise in nucleotide selection are corrected in a second process called proofreading. When a DNA polymerase inserts an incorrect nucleotide into the growing strand, the 3'-OH group of the mispaired nucleotide is not correctly positioned for accepting the next nucleotide. The incorrect positioning stalls the poly-

Two units of DNA polymerase III

Leading strand

Lagging strand

Helicase-primase complex

DNA gyrase Third Primer

Single-strand-binding proteins a

The lagging strand loops around so that 5'—3' synthesis can take place on both antiparallel strands.

First primer

Second primer Third primer

0 0

Post a comment