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Lewis: Human Genetics: Concepts and Applications, Ninth Edition

III. DNA and Chromosomes

9. DNA Structure and Replication

The McGrawHill Companies, 2010

Enzymes in DNA replication

S phase 3
C T A G T A G C A T G A C G

5 1 Parent DNA molecule. 1

Helicase unwinds parental double helix.

Binding proteins stabilize separate strands.

Primase adds short primer to template strand.

2 Parental strands unwind and separate at several points.

DNA polymerase binds nucleotides to form new strands.

Ligase joins Okazaki fragments and seals other nicks in sugarphosphate backbone.

3
G T A G C A T

G A G C A T

G A

Figure 9.15

Overview of DNA replication.

After experiments demonstrated the semiconservative nature of DNA replication, the next challenge was to decipher the steps of the process.

Steps of DNA Replication

DNA replication occurs during S phase of the cell cycle (see figure 2.13). When DNA replicates, it unwinds, breaks, builds a new nucleotide chain, and mends (figure 9.15). Enzymes called helicases unwind and hold apart replicating DNA, enabling other enzymes to guide the assembly of a new DNA strand. Human DNA replicates about 50 bases per second. To get the job done, a human chromosome replicates simultaneously at hundreds of points along its length, and the pieces join. A site where DNA is locally opened, resembling a fork, is called a replication fork. DNA replication begins when a helicase breaks the hydrogen bonds that connect a base pair (figure 9.16). Binding proteins hold the two strands apart. Another enzyme, primase, then attracts complementary RNA nucleotides to build a short piece of RNA, called an RNA primer, at the start of each segment of DNA to be replicated. The RNA primer is required because the major replication enzyme, DNA polymerase (DNAP), can only add bases to an existing nucleic acid strand. (A polymerase is an enzyme that builds a polymer, which is a chain of chemical building blocks.) Next, the RNA primer attracts DNAP, which brings in DNA nucleotides complementary to the exposed bases on the parental strand; this strand serves as a mold, or template. New bases are added one at a time, starting at the RNA primer.
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Part 3 DNA and Chromosomes
6 6 Continuous strand synthesis continues in a 5 to 3 direction. 5 3 7 Discontinuous synthesis produces Okazaki fragments on the 5 to 3 template. 5 7 3 5 3 5 Okazaki fragment 3 3 5

G T

C C

3
3 Each parental strand provides a template for DNA polymerase to bind complementary bases, A with T and G with C. 4 Sugar-phosphate backbones of daughter strands close.

1 Helicase binds to origin and separates strands. 5 3 2 Binding proteins keep strands apart. 3 Primase makes a short stretch of RNA on the DNA template.

3 1

5 2 3

3 5

4 DNA polymerase adds DNA nucleotides to the RNA primer. Overall direction of replication 5 3 5 DNA polymerase proofreading activity checks and replaces incorrect bases.

4 3 5

5 5 3 5

3 3 5 3 5 8 Enzymes remove RNA primers. Ligase seals sugar-phosphate backbone. 3 5 8 3 5 5

Figure 9.16

Activities at the replication fork. DNA replication takes many steps.

Lewis: Human Genetics: Concepts and Applications, Ninth Edition

III. DNA and Chromosomes

9. DNA Structure and Replication

The McGrawHill Companies, 2010

181

The new DNA strand grows as hydrogen bonds form between the complementary bases. The nucleotides are abundant in cells, and are synthesized from dietary nutrients. DNAP works directionally, adding new nucleotides to the exposed 3 end of the sugar in the growing strand. Overall, replication proceeds in a 5 to 3 direction, because this is the only chemical configuration in which DNAP can add bases. How can the growing fork proceed in one direction, when both parental strands must be replicated? The answer is that on at least one strand, replication is discontinuous. It is accomplished in small pieces from the inner part of the fork outward, in a pattern similar to backstitching. Next, an enzyme called a ligase then seals the sugar-phosphate backbones of the pieces, building the new strand. These pieces, up to 150 nucleotides long, are called Okazaki fragments, after their discoverer (see figure 9.16). DNA polymerase also proofreads as it goes, excising mismatched bases and inserting correct ones. It also removes the RNA primer and replaces it with the correct DNA bases. Yet another enzyme, called an annealing helicase, rewinds any sections of the DNA molecule that remain unwound. Finally, ligases seal the entire sugar-phosphate backbone. Ligase comes from a Latin word meaning to tie. As a human body grows to 100 trillion or so cells, DNA replication occurs about 100 quadrillion times. Some of the

DNA sequence encodes protein, but most does not. The genome is in this sense a little like a booklet describing the parts of a machine, such as a car, that comes with a much more extensive manual that explains the details of keeping it in working order. The next chapters in this part of the book explain how the human instruction manual is accessed, and explores some of the nuances of its use. The Bioethics: Choices for the Future box discusses a controversial use of DNA testing.