Figure 5–26 Methylation of the E. coli replication origin creates a refractory period for DNA initiation. DNA methylation occurs at GATC sequences, 11 of which are found in the origin of replication (spanning approximately 250 nucleotide pairs). In its hemimethylated state, the origin of replication is bound by an inhibitor protein (Seq A, not shown), which blocks the ability of the initiator proteins to unwind the origin DNA. Eventually (about 15 minutes after replication is initiated), the hemimethylated origins become fully methylated by a DNA methylase enzyme; Seq A then dissociates.
A single enzyme, the Dam methylase, is responsible for methylating all E. coli GATC sequences. A lag in methylation after the replication of GATC sequences is also used by the E. coli mismatch proofreading system to distinguish the newly synthesized DNA strand from the parental DNA strand; in that case, the relevant GATC sequences are scattered throughout the chromosome, and they are not bound by Seq A.
Figure 5–27 The experiments that demonstrated the pattern in which replication forks are formed and move on eukaryotic chromosomes. The new DNA made in human cells in culture was labeled briefly with a pulse of highly radioactive thymidine (3H-thymidine).
(A) In this experiment, the cells were lysed, and the DNA was stretched out on a glass slide that was subsequently covered with a photographic emulsion. After several months, the emulsion was developed, revealing a line of silver grains over the radioactive DNA. The brown DNA in this figure is shown only to help with the interpretation of the autoradiograph; the unlabeled DNA is invisible in such experiments. (B) This experiment was the same except that a further incubation in unlabeled medium allowed additional DNA, with a lower level of radioactivity, to be replicated. The pairs of dark tracks in (B) were found to have silver grains tapering off in opposite directions, demonstrating bidirectional fork movement from a central replication origin where a replication bubble forms (see Figure 5–23). A replication fork is thought to stop only when it encounters a replication fork moving in the opposite direction or when it reaches the end of the chromosome; in this way, all the DNA is eventually replicated.
258 Chapter 5:  DNA Replication, Repair, and Recombination culture of cells arrested before DNA replication begins allow replication to begin fragment DNA, separate strands, and fuorescently label genes are being expressed. The excess origins also provide “backups” in case a primary origin fails. (3) As in bacteria, replication forks are formed in pairs and create a replication bubble as they move in opposite directions away from a common point of origin, stopping only when they collide head-on with a replication fork moving in the opposite direction or when they reach a chromosome end. In this way, many replication forks operate independently on each chromosome and yet form two complete daughter DNA helices.
In Eukaryotes, DNA Replication Takes Place During Only One Part of the Cell Cycle
When growing rapidly, bacteria replicate their DNA nearly continuously. In contrast, DNA replication in most eukaryotic cells occurs only during a specific part of the cell-division cycle, called the DNA synthesis phase or S phase (Figure 5–29). In a mammalian cell, the S phase typically lasts for about 8 hours; in simpler eukaryotic cells such as yeasts, the S phase can be as short as 40 minutes. By its end, each chromosome has been replicated to produce two complete copies, which remain joined together at their centromeres until the M phase (M for mitosis), which soon follows. In Chapter 17, we describe the control system that runs the cell cycle, and we explain why entry into each phase of the cycle requires the cell to have successfully completed the previous phase.
In the following sections, we explore how chromosome replication is coordinated within the S phase of the cell cycle.
Different Regions on the Same Chromosome Replicate at Distinct Times in S Phase