Figure 5–25 The proteins that initiate DNA replication in bacteria. The mechanism shown was established by studies in vitro with mixtures of highly purified proteins. For E. coli DNA replication, the major initiator protein, the helicase, and the primase are the dnaA, dnaB, and dnaG proteins, respectively. In the first step, several molecules of the initiator protein bind to specific DNA sequences at the replication origin and destabilize the double helix by forming a compact structure in which the DNA is tightly wrapped around the protein. Next, two helicases are brought in by helicaseloading proteins (the dnaC proteins), which inhibit the helicases until they are properly loaded at the replication origin. Helicase-loading proteins prevent the replicative DNA helices from inappropriately entering other single-strand stretches of DNA in the bacterial genome. Aided by single-strand binding protein (not shown), the loaded helicases open up the DNA, thereby enabling primases to enter and synthesize initial primers. In subsequent steps, two complete replication forks are assembled at the origin and move off in opposite directions. The initiator proteins are displaced as the left-hand fork moves through them (not shown).
fully methylated hemimethylated origins are origin resistant to initiation initiation occurs if suffcient origins become fully resources are available to complete methylated, making them a round of DNA replication again competent for initiation
In this way, both the rate and the direction of replication-fork movement can be determined (Figure 5–27). From the rate at which tracks of replicated DNA increase in length with increasing labeling time, the eukaryotic replication forks are estimated to travel at about 50 nucleotides per second. This is approximately twentyfold slower than the rate at which bacterial replication forks move, possibly reflecting the increased difficulty of replicating DNA that is packaged tightly in chromatin.
An average-size human chromosome contains a single linear DNA molecule of about 150 million nucleotide pairs. It would take 0.02 seconds/nucleotide × 150 × 106 nucleotides = 3.0 × 106 seconds (about 35 days) to replicate such a DNA molecule from end to end with a single replication fork moving at a rate of 50 nucleotides per second. As expected, therefore, the autoradiographic experiments just described reveal that many forks, belonging to separate replication bubbles, are moving simultaneously on each eukaryotic chromosome.
Much faster and more sophisticated methods now exist for monitoring DNA replication initiation and tracking the movement of DNA replication forks across whole genomes. One approach uses DNA microarrays—grids the size of a postage stamp studded with hundreds of thousands of fragments of known DNA sequence. As we will see in detail in Chapter 8, each different DNA fragment is placed at a unique position on the microarray, and whole genomes can thereby be represented in an orderly manner. If a DNA sample from a group of replicating cells is broken up and hybridized to a microarray representing that organism’s genome, the amount of each DNA sequence can be determined. Because a segment of a genome that has been replicated will contain twice as much DNA as an unreplicated segment, replication-fork initiation and fork movement can be accurately monitored across an entire genome (Figure 5–28).
Experiments of this type have shown the following: (1) Approximately 30,000– 50,000 origins of replication are used each time a human cell divides. (2) The human genome has many more (perhaps tenfold more) potential origins than this, and different cell types use different sets of origins. This may allow a cell to coordinate its active origins with other features of its chromosomes such as which