in DNA are single-ring structures called pyrimidines and the other two (A and G) are double-ring struc­tures called purines. The charge interactions between purines and pyrimidines allow them to form weak hydrogen bonds (FIG. 7-7). Chemically the most sta­ble hydrogen bonding occurs when guanine forms three hydrogen bonds with cytosine and when ade­nine forms two hydrogen bonds with thymine. The proper alignment to form these hydrogen bonds oc­curs only when the sugar-phosphate backbones of the two DNA chains run in opposing directions and are twisted together to form the double helix.

DNA, which stores and transmits cellular hereditary

Information, is a double helical molecule.

The hydrogen bonding of A to T and C to G is called base pairing. It is this complementarity that establishes the basis for the double helical arrange­ment of DNA and for the accurate replication of the DNA macromolecule. This is essential for passage of hereditary information from one generation to the next. It also means that in the double helical DNA molecule, the amount of adenine is always the same as the amount of thymine, and the amount of gua­nine is always the same as the amount of cytosine (A = T and G = C).

FIG. 7-7 Hydrogen bonding occurs between nucleotide base pairs. Adenine forms two hydrogen bonds with thymine. Guanine forms three hydrogen bonds with cyto­sine.

Base pairing occurs between complementary nu­cleotides—adenine pairs with thymine and guanine pairs with cytosine.

REPLICATION OF DNA

When a cell divides, its hereditary information is passed to the next generation. Replication of the hereditary information involves synthesizing new DNA molecules that have the same nucleotide se­quences as those of the parental organism. The trans­fer of hereditary information is possible because DNA has a unique chemical structure in which the two chains of the DNA double helix are complemen­tary in nucleotide sequence. Wherever a G is found in one chain, a C is found in the other, and wherever a T is present in one chain, its complementary chain will have an A. A nucleotide sequence of ATCG in one chain has a corresponding sequence of TAGC in the other chain. The nucleotide sequence in one chain specifies the sequence in the other. The information in DNA is, thus, accurately replicated so that an exact copy is passed from one generation to the next.

The order of nucleotides in each chain of a double helical DNA molecule specifies the order of nu­cleotides in the new complementary chains.

Semiconservative DNA Replication

The process by which a double helical DNA molecule is copied to form a duplicate DNA macromolecule is

called semiconservative replication. It is so named because during replication each of the chains of nucleotides in the DNA being replicated remains intact, The two chains of nucleotides in the double-stranded DNA molecule are conserved—and a new, comple­mentary chain is assembled for each one. Each of the conserved parental DNA chains serves as the tem­plate that specifies the sequence of nucleotides in the newly synthesized strands.

Semiconservative replication was demonstrated experimentally by Matthew Meselson and Franklin Stahl at the California Institute of Technology in 1958 (FIG. 7-8). They grew a culture of Escherichia coli in a medium in which the sole source of nitrogen was the heavy isotope ¹⁵N. The heavy nitrogen was incorpo­rated into the nucleotides of DNA during bacterial reproduction, so that the DNA of these bacteria be­came heavier than usual. They then transferred these bacteria to a medium containing the normal lighter isotope ¹⁴N. At various time intervals they collected cells and analyzed the DNA to determine if it was "heavy" (¹⁵N label), light (¹⁴N label), or intermediate (mixture of ¹⁵N and ¹⁴N label). For these analyses they used an ultracentrifuge—an instrument that spins its contents at high speed—which caused materials tot separate out according to their different densities.

Denser molecules move farther than lighter mole­cules in cesium chloride density gradient centrifuga­tion, so DNA containing ¹⁵N moves a greater distance than DNA containing only ¹⁴N. The movement is such that bands of DNA can be distinguished corre­sponding to light, heavy, and intermediate DNA.

Initially Meselson and Stahl detected only one band. This band corresponded to heavy DNA in which both chains of the DNA contained the ¹⁵N la­bel. After sufficient time for one complete round of DNA replication, again only one band of DNA was detected, but now the band was at an intermediate level between all-light isotope and all-heavy isotope DNA. This intermediate band was exactly what was predicted by the hypothesis that DNA replication is semiconservative. Each DNA double helix had one chain from the parental DNA that contained the heavy ¹⁵N isotope and one newly synthesized chain that contained only the light ¹⁴N isotope. Also as pre­dicted, after sufficient time for a second round of DNA replication, Meselson and Stahl observed two bands of DNA, one intermediate and the other light. This occurred because when the intermediate DNA containing one light and one heavy chain replicated, it contributed one heavy chain to form another inter­mediate DNA macromolecule and one light chain to form a new all-light DNA macromolecule. This ex­periment confirmed that DNA replication is semi­conservative as suggested by the Watson-Crick model of the DNA double helix.

DNA replication is semiconservative, producing two "holf-old, half-new" DNA macromolecules every time the DNA is duplicated.

Steps in DNA Replication

Unwinding the DNA Double Helix—Replication Forks

The first step in semiconservative DNA replication is to pull apart a portion of the DNA helix. This enables each of the chains to act as a template (pattern) to di­rect the synthesis of a new complementary chain of nucleotides. This can occur because hydrogen bonds are relatively weak. Thus the two chains can separate without breaking apart the covalently linked nu­cleotides of the chains, which would destroy the in­formation encoded within them. This establishes the basis for one chain serving as a template for the syn­thesis of a new chain of DNA with a sequence of nucleotides that is exactly complementary.

The chains do not entirely separate before DNA replication. Rather, a localized region of the DNA un­winds because the two parental DNA chains are pulled apart by specific enzymes. This creates a re­gion of two single strands and provides space for in­dividual nucleotides to align opposite their comple­mentary bases for the synthesis of new chains. This region of localized DNA synthesis is called a replica­tion fork (FIG. 7-9, p. 201). At the replication fork, en­zymes link nucleotides to form a new DNA strand that is complementary to the original template DNA.

The DNA double helix unwinds to form a replication fork where DNA synthesis occurs.

In eukaryotic cells, multiple replication forks form at different locations. Simultaneous synthesis of dif­ferent portions of the DNA is thus made possible. In a bacterial cell, DNA replication is initiated at only

REPLICATION OF DNA 199