whwytrirightIn dispersive replication, the density of the new DNAwould have been half that of parental DNA.The Meselson-Stahl experiment, called by some scientistsamong the most elegant ever done by biologists, was an ex-cellent example of the scientific method. It began with threehypotheses—the three models of DNA replication—and wasdesigned so that the results could differentiate between them.Semiconservative DNA replication in the cell involves a num-ber of different enzymes and other proteins. It takes place intwo steps:whwytrirightThe DNA is unwound to separate the two templatestrands and make them available for base pairing.whwytrirightNew nucleotides are linked by covalent bonding to eachgrowing new strand in a sequence determined by com-plementary base pairing with the bases on the templatestrand.A key observation of virtually all DNA replication is thatnucleotides are always added to the growing strand at the 3′end—the end at which the DNA strand has a free hydroxyl (—OH)group on the 3′carbon of its terminal deoxyribose (Figure11.10). The three phosphate groups in a deoxyribonucleosidetriphosphate are attached to the 5′position of the sugar (seeFigure 11.7). So when a new nucleotide is added to DNA, itcan attach only to the 3′end.When DNA polymerase brings a deoxyribonucleosidetriphosphate with the appropriate base to the 3′end of agrowing chain, the free hydroxyl group on the chain reactswith one of the substrate’s phosphate groups. As this hap-pens, the bond linking the terminal two phosphate groups tothe rest of the deoxyribonucleoside triphosphate breaks, andstored energy is released as the phosphate groups separatefrom the molecule. The resultingpyrophosphate ion, consist-ing of the two terminal phosphate groups, also hydrolyzes,forming two separate phosphate ions and in the process re-leasing additional free energy. The phosphate group still onthe nucleotide becomes part of the sugar–phosphate back-bone of the growing DNA molecule.DNA is replicated through the interaction of the templateDNA with a huge protein complex called thereplicationcomplex, which catalyzes the reactions involved. All chro-mosomes have at least one base sequence, called theoriginof replication, to which this replication complex initiallybinds. DNA replicatesin both directionsfrom the origin ofreplication, forming tworeplication forks(Figure 11.11).Both of the separated strands of the parent molecule act astemplates simultaneously, and the formation of the newstrands is guided by complementary base pairing.Until recently, DNA replication was depicted as a loco-motive (the replication complex) moving along a railroadtrack (the DNA) (Figure 11.11a). The current view is that thismodel may not be correct. Instead, the replication complexseems to be stationary, attached to nuclear structures, and it isthe DNA that moves, essentially threading through the com-plex as single strands and emerging as double strands (Fig-ure 11.11b). During S phase in eukaryotes, there are about 100replication complexes, and each of them contains as many as300 individual replication forks. All replication complexescontain several proteins with different roles in DNA replica-tion; we will describe these proteins as we examine the stepsof the process.
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