分子生物学Chapter 4 DNA Replication.doc

Chapter 4 DNA Replication IntroductionThe Watson-Crick duplex structure of DNA immediately suggested how genetic material was duplicated from one generation to the next. The realization that bacterial genomes and eukaryotic chromosomes consist of single DNA molecules millimeters to centimeters in length raised a host of structural and biochemical questions about DNA replication. How does replication begin? Which enzymes take part in DNA synthesis, and what are their functions? How does duplication of the long helical duplex occur without the strands becoming tangled?As we will see, to carry out DNA replication, all cells use the same kinds of enzymes, including DNA polymerases, which assemble deoxyribonucleotides into a new strand; helicases, which unwind duplex DNA; single-stranded DNA-binding proteins; and exonucleases. Bacteria and yeast contain three different DNA polymerases, and mammalian cells contain five. Each type of polymerase has unique functions during DNA replication and repair. Additionally, some of the enzymes that cut and repair damaged DNA also participate in genetic recombination. Another common feature of the three processes discussed in this chapter is involvement of large multisubunit complexes, each containing many enzymes and structural proteins. These molecular machines have evolved to ensure that DNA replication, repair, and recombination occur very rapidly and with exquisite precision.Autoradiographic analyses carried out in the early 1960s on whole replicating chromosomes labeled with a short pulse of the radioactive DNA precursor 3H-thymidine revealed a localized region of replication that moves along the parental DNA double helix. Because of its Y-shaped structure, this active region is called a DNA replication fork. At a replication fork the DNA of both new daughter strands is synthesized by a multienzyme complex that contains the DNA polymerase.Figure 4-1 The replication fork is the region of DNA in which there is a transition from the unwound parental duplex to the newly replicated daughter duplexes. (Animated Figure)1. General Features of Chromosomal Replication One Sentence Statement:DNA replication proceeds Sequentially and Semi-Discontinuously from one or more Unique, Heritable Origin(s) at one (UniDirectional) or two (BiDirectional) replication Fork(s) in an overall Semiconservative replication mode to a Terminus, followed by Daughter Chromosome separation and Segregation.We first consider general features of DNA replication: the semiconservative and bidirectional growth of new strands from a common site.1.1 Semiconservative Mechanism of DNA Replication The base-pairing principle inherent in the Watson-Crick model suggested that the two new DNA strands are copied from the two old strands. Although this mechanism provides for exact copying of genetic information, it raised a new question: Is replication a conservative or semiconservative process? In the first mechanism, the two new strands form a new duplex and the old duplex remains intact, whereas in the second mechanism, each old strand becomes paired with a new strand copied from it (Figure 4-2).The parental duplex is replicated to form two daughter duplexes, each of which consists of one parental strand and one (newly synthesized) daughter strand. The unit conserved from one generation to the next is one of the two individual strands comprising the parental duplex. This behavior is called semiconservative replicationThe first definitive evidence supporting a semiconservative mechanism came from a classic experiment by M. Meselson and W. F. Stahl. E. coli cells initially were grown in a medium containing ammonium salts prepared with “heavy” nitrogen (15N) until all the cellular DNA contained the isotope. The cells were then transferred to a medium containing the normal “light” isotope (14N), and samples were removed periodically from the cultures. The DNA in each sample was analyzed by density-gradient equilibrium centrifugation, which can separate heavy-heavy (H-H), light-light (L-L), and heavy-light (H-L) duplexes into distinct bands. The actual banding patterns observed were consistent with semiconservative replication and inconsistent with conservative replication. Subsequent experiments of a different design with cultured plant cells demonstrated for the first time semiconservative DNA replication in eukaryotic chromosomes. Apparently all cellular DNA in both prokaryotic and eukaryotic cells is replicated by a semiconservative mechanism. For further information on these early experiments on semiconservative replication.Figure 4-2. Conservative and semiconservative mechanisms of DNA replication differ in whether the newly synthesized strands pair with each other (conservative) or with an old strand (semiconservative).All available evidence supports semiconservative replication in both prokaryotic and eukaryotic1.2 Bidirectional ReplicationSeveral possible molecular mechanisms of DNA strand growth would result in semiconservative DNA replication. In one of the simplest possibilities, one new strand derives from one origin and the other new strand derives from another origin. Only one strand of the duplex grows at each growing point. In this mechanism, which operates in linear DNA viruses such as adenovirus, the ends of the DNA molecules serve as fixed sites for the initiation and termination of replication. A second possibility entails one origin and one growing fork (the point where DNA replication occurs), which moves along the DNA in one direction with both strands of DNA being copied (Figure 4-3b). Certain bacterial plasmids replicate in this manner. A third possibility is that synthesis might start at a single origin and proceed in both directions, so that both strands are copied at each of two growing forks (Figure 4-3c). The available evidence suggests that the third alternative is generally employed by prokaryotic and eukaryotic cells: that is, DNA replication proceeds bidirectionally from a given starting site, with both strands being copied at each fork. Thus two growing forks emerge from a single origin site. Figure 4-3. Three mechanisms of DNA strand growth that are consistent with semiconservative replication. The third mechanism bidirectional growth of both strands from a single origin appears to be the most common in both eukaryotes and prokaryotes.Evidence for Bidirectional Replication The first experimental support for bidirectional replication in eukaryotic cells was obtained by fiber autoradiography of labeled DNA molecules from cultured mammalian cells. Such studies have revealed clusters of active replicons, each of which contains two growing forks moving away from a central origin, thus providing unambiguous evidence of bi-directional growth. Most cellular DNA and many viral DNA molecules replicate bidirectionally. Such viruses serve as excellent models for the study of cellular DNA replication.If DNA from replicating eukaryotic cells is extracted and examined by electron microscopy, so-called replication “bubbles,” or “eyes,” extending from multiple replication origins are visible. Although such micrographs do not constitute conclusive evidence for unidirectional or bidirectional fork movement, electron-microscope studies of bubbles in viral DNA have provided evidence for bidirectional replication. If circular viral DNA molecules at different stages of replication are cut with a restriction endonuclease that recognizes a single site, the positions at the center of the replication bubble with respect to the restriction site can be determined. The most common result from such analyses is a series of ever larger bubbles whose centers map to the same site, indicating bidirectional replication of both DNA strands from that site. Thus both fiber autoradiography and electron microscopy have indicated that bidirectional DNA replication is the general rule. In the circular DNA molecules present in bacteria, plasmids, and some viruses, one origin often suffices, and the two resulting growing forks merge on the opposite side of the circle to complete replication.Theta Structures:Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separate from a cells chromosomal DNA. These extrachromosomal DNAs, which occur naturally in bacteria, yeast, and some higher eukaryotic cells, exist in a parasitic or symbiotic relationship with their host cell. Plasmids range in size from a few thousand base pairs to more than 100 kilobases (kb).Figure 4-4. Circle DNA replication. The parental strands are shown in blue, and newly synthesized daughter strands are shown in red. The short segments represent the AT and GC base pairs connecting the complementary strands. Once DNA replication is initiated at the origin (ORI), it continues in both directions around the circular molecule until the advancing growing forks merge and two daughter molecules are produced. The origin is the only specific nucleotide sequence required for replication of the entire circular DNA molecule1.3 DNA Replication originv Way should the fragment of DNA be inserted into a plasmid vector which is subsequently replicated inside a bacterial host 生长快、耐不良环境、肉质好的转基因鱼 However, the long linear chromosomes of eukaryotes contain multiple origins; the two growing forks from a particular origin continue to advance until they meet the advancing growing forks from neighboring origins. Each region served by one DNA origin is called a replicon.a stretch of DNA that is necessary and sufficient for replication of a circular DNA molecule, usually a plasmid or virus, in an appropriate host cell. In yeast, this definition has been refined to include sequences that direct replication once per S phase, the period of the cell cycle in which chromosomal duplication takes placereplicon: unit of the genome which is replicated(self-replicated)A replicon contains:an origin of replication: the point at which DNA replication initiatesDNA to be replicated: may be as large as an entire chromosome or as small as several kilobasesterminators (maybe)E. coli: contains a single origin of replication on its circular chromosome (4.2 Mb); fork moves at 50,000 bp/minMouse: contains 25,000 replicons, 150 kb each; distributed between multiple linear chromosomes; forks move at 2,200 bp/minNumber of Growing Forks and Their Rate of Movement In E. coli cells, it takes about 42 minutes to replicate the single circular chromosome, which contains exactly 4,639,221 base pairs and is about 1.4 mm in length. Since the chromosome is duplicated from one origin by two growing forks, we can calculate that the rate of fork movement is about 1000 base pairs per second per fork. The rate of growing fork movement determined from fiber autoradiography of E. coli cells labeled for various times agrees with this calculated value, indicating that the fiber-labeling technique can provide a reasonable estimate of the rate of growing fork movement in vivo.The rate of fork movement in human cells, based on fiber-labeling experiments, is only about 100 base pairs per second per fork. The entire human genome of 3 109 base pairs replicates in 8 hours; in this time, one fork theoretically could replicate 3 106 base pairs, suggesting that the human genome must contain a minimum of 1000 growing forks. However, fiber autoradiography and electron microscopy indicate that growing forks are spaced closer than 3 106 base pairs apart. A more likely estimate is that the human genome contains 10,000 100,000 replicons, each of which is actively replicating for only part of the 8 hours required for replication of the entire genome. Multiple eukaryotic repliconsPerhaps the most important decision every cell has to make is whether, and when, to replicate its DNA. DNA replication, like RNA synthesis and many other biological processes, is controlled at the initiation step. Such control would be most efficient if there are specific sites on chromosomes at which DNA replication always begins in vivo. As noted already, electron-microscope studies have shown that animal viruses have replication bubbles whose centers are always in the same approximate site. Similar results have been obtained with circular bacterial and plant viruses and for bacterial, yeast, and mammalian plasmids. More detailed molecular studies indicate that replication of these DNAs actually begins at a defined sequence of base pairs near the center of these bubbles, called the replication origin. Genetic and recombinant DNA experiments provide another way to define a replication origin experimentally as a stretch of DNA that is necessary and sufficient for replication of a circular DNA molecule, usually a plasmid or virus, in an appropriate host cell. In yeast, this definition has been refined to include sequences that direct replication once per S phase, the period of the cell cycle in which chromosomal duplication takes place. We discuss three types of replication origins to illustrate some general conclusions about their nature: the E. coli oriC, yeast autonomously replicating sequences, and the simian virus 40 (SV40) origin. The detailed knowledge now available about the proteins required to start replication at the E. coli origin and the accumulating information about other origins and their use in vitro all suggest that most cellular DNA replication begins at specific sequences, possibly using similar mechanisms.1.3.1. E. coli Replication Origin The E. coli replication origin oriC is an 240-bp DNA segment present at the start site for replication of E. coli chromosomal DNA. Plasmids or any other circular DNA containing oriC are capable of independent and controlled replication in E. coli cells. Comparison of oriC with the origins of five other bacterial species including the distant species Vibrio harveyi, a marine bacterium, revealed that all contain repetitive 9-bp and AT-rich 13-bp sequences, referred to as 9-mers (dnaA boxes) and 13-mers, respectively (Figure 4-5). As we will see later, these are binding sites for the DnaA protein that initiates replication. In addition, the E. coli genome contains a segment of DNA with a relatively high A+T content adjacent to oriC; this sequence appears to be important in facilitating local melting of the duplex to reveal the two single-stranded DNA segments onto which the DNA replication machinery is loaded.Figure 4-5. Consensus sequence of the minimal bacterial replication origin based on analyses of genomes from six bacterial species. Similar sequences constitute each origin; the 13-bp repetitive sequences (orange) are rich in adenine and thymine residues. The 9-bp repetitive sequences (brown) exist in both orientations; that is, the lower-right sequence, read right to left, is the same as that of the upper-left sequence, read left to right. These sequences are referred to as 13-mers and 9-mers, respectively. Indicated nucleotide position numbers are arbitrary. See J. Zyskind et al., 1983, Proc. Natl. Acad. Sci. USA 80:1164.After E. coli DNA replication has initiated, the replication origins in the two daughter DNA duplexes become linked to specific proteins at different points on the plasma membrane. As the cell wall that divides the cell in two forms, this linkage ensures that one of the new DNA duplexes is delivered to each daughter cell. There is no visible condensation and decondensation of the DNA in bacterial cells, as there is in eukaryotic cells during mitosis. 1.3.2 Yeast Autonomously Replicating Sequences Each yeast chromosome, like all eukaryotic chromosomes, has multiple origins of replication. Cloning experiments indicate that about 400 origins exist in the 17 chromosomes of S. cerevisiae; more than a dozen of these have been characterized in detail. Each yeast origin sequence, called an autonomously replicating sequence (ARS), confers on a plasmid the ability to replicate in yeast and is a required element in yeast artificial chromosomes. Detailed mutational analysis of one 180-bp ARS called ARS1 revealed only one essential element, a 15-bp segment, designated element A, stretching from position 114 through 128. Three other short segments the B1, B2, and B3 elements increase the efficiency of ARS1 functioning. Comparison of the sequences required for functioning of many different DNA segments that act as ARSs led to recognition of an 11-bp consensus sequence:Element A in ARS1 is identical at 10 of 11 positions of this consensus sequence, and element B2 at 9 of 11. DNase footprinting (see Figure 4-6) has shown that a complex of six different proteins called the ORC (origin-recognition complex) binds specifically to element A in ARS1 in an ATP-dependent manner. This complex also binds specifically to other ARSs tested. The ORC remains bound to an ARS throughout the cell cycle and during replication becomes associated with other proteins, an event that apparently triggers initiation of DNA synthesis. Yeast cells with mutations in any of these six ORC proteins are defective in DNA replication. All eukaryotic cells express homologs of these proteins, attesting to their importance in initiation of DNA replication. Figure 4-5-2. Sequences of bacterial and yeast replication origins. (a) The bacterial replication origin contains three homologous 13-mers and four homologous 9-mers. (b) The yeast replication origin is called autonomously replicating sequence (ARS). The A region is absolutely necessary, while B1, B2, and B3 can increase the replication efficiency.Figure 4-6. DNase I footprinting, a common technique for identifying protein-binding sites in DNA. (Top) A DNA fragment is labeled at one end with 32P (red dot) as in the Maxam-Gilber