Читать книгу Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies - Kenneth N Kreuzer - Страница 12

The next several chapters will consider detailed aspects of the proteins and reactions involved in DNA replication. To set the stage for these chapters, let us first consider the very basic functions needed for replication — the basic reactions that must occur for successful genome duplication.

The heart of any DNA replication reaction is the ability to polymerize the deoxynucleoside monophosphates in the new daughter strand, a reaction catalyzed by enzymes called “DNA polymerases.” DNA polymerases use the rules of base pairing to accurately select which deoxynucleotide to insert into a growing chain (Figure 1.4A). As shown in the figure, the two parental strands have distinct roles — the chain to which residues are added is called the “primer” and the chain used for testing the base pair is called the “template,” since it is the template for the information in the newly synthesized region.

Figure 1.4.(Figure on Facing Page) The basic synthetic step in DNA synthesis. The reaction catalyzed by DNA polymerases involves the addition, sequentially, of single nucleotide residues to a DNA chain. The new residue is always added to the free 3′ end of a preexisting primer, and hence the direction of chain growth is 5′ to 3′. DNA polymerase nearly always inserts the nucleotide which correctly base pairs with the opposing base on the template strand, in this case, a C residue opposite a template G residue (panel A). The precursors for DNA synthesis are nucleoside triphosphates, with monophosphate incorporated into the DNA and pyrophosphate released. Panel B shows the reaction in more detail, highlighting the chemistry of nucleotide addition within the dotted boxes. The 3′-OH group on the primer chain performs a nucleophilic attack on the first (α) phosphate of the incoming nucleoside triphosphate, releasing pyrophosphate, and cementing the bridge (the phosphodiester bond) between the newly added residue and the growing primer chain. Notice that the polymer product of the reaction on the right has a free 3′-OH group on the newly added residue, allowing this 3′-OH group to serve as the attacking group for the next nucleotide addition.

The substrates for this extension reaction are nucleoside triphosphates, with the triphosphate attached to the 5′-C of the deoxyribose. The reaction involves a phosphoryl transfer reaction in which the 3′-OH group at the primer terminus engages in a nucleophilic attack on the first phosphate group (α) of the high-energy nucleoside triphosphate (Figure 1.4B). The reaction results in cleavage of the linkage between the first and second (β) phosphate groups, with the 3′-OH of the primer becoming linked to the α phosphate of the incoming residue. The β and γ phosphates are released in the reaction, still linked to each other as pyrophosphate. Note that the primer has now been extended by one residue, and the 3′-OH group of the newly added residue is now the new 3′ end of the growing primer strand. The next cycle of nucleotide addition will utilize this 3′-OH group to direct the nucleophilic attack of the new nucleoside triphosphate in the polymerase reaction. Successive rounds of the addition result in extensive chain growth in the 5′ to 3′ direction.

Nearly all DNA polymerases require a preexisting primer and extend new chains in the 5′ to 3′ direction (see Section 4.9 for the sole exception). The consequences of these characteristics will be evident at many points in this book. The most immediate consequence relates to the difference in replicating the two strands. As the DNA replication machinery replicates a parental duplex from one location to another, one of the strands can be synthesized continuously in the 5′ to 3′ direction, potentially by a single DNA polymerase molecule that continuously adds residues, and this strand is called the “leading strand.” The other strand, however, requires a DNA polymerase that travels in the opposite direction with respect to its template strand (Figure 1.5). Since the synthesis of this strand is somewhat delayed compared to the leading strand, it is referred to as the “lagging strand.” Note also that the lagging strand cannot be made in one continuous process (unless the process is started only after the entire leading strand is completed). Instead, the lagging strand is synthesized in short pieces, which are joined together after synthesis to form the complete daughter strand. These short pieces are called “Okazaki fragments,” after the scientist who discovered them in the 1960s.³

Figure 1.5.Simplified schematic of a replication fork. The new leading strand can be synthesized continuously due to the 5′ to 3′ directionality of DNA polymerases, but the new lagging strand must be synthesized discontinuously in short Okazaki fragments, which are later joined together. The major protein players at the replication fork include DNA polymerase (dark blue), helicase (orange), primase (light blue), and ssDNA-binding protein (green). See text for further discussion.

While one particular protein species carries the active site for DNA synthesis and can rightly be called the DNA polymerase itself, every replicative DNA polymerase acts within a complex of multiple protein species. The additional protein subunits greatly modulate the activity of the polymerase, for example, allowing the different behavior of DNA polymerase on the leading and lagging strand.

As mentioned above, DNA polymerases generally require a preexisting primer to add deoxynucleotide residues. How then can DNA synthesis be started at the locations where leading-strand synthesis begins, and the multiple locations where Okazaki fragments begin? It turns out that RNA polymerases do not have this requirement for a preexisting primer, even though they share many of the other characteristics of DNA polymerases (e.g., synthesizing product in the 5′ to 3′ direction, using the rules of base pairing, using triphosphate nucleotide substrates, releasing pyrophosphate, etc.). RNA polymerases involved in transcription begin their RNA synthesis at sequence elements called promoters, linking two nucleoside triphosphates in the first step (Figure 1.6). From there on, additional nucleoside monophosphates are added in a reaction essentially identical to that in DNA synthesis mentioned above.

Getting back to DNA replication, the second major function needed for the functional replication machinery is a special form of RNA polymerase that provides short RNA primers, which are extended by the DNA polymerases (Figure 1.5). In most systems, these primases provide the primer for the leading-strand polymerase reaction at the sites where replication begins, called replication origins. They also function repeatedly during lagging-strand synthesis to prime the synthesis of every Okazaki fragment. Note also that these short stretches of RNA residues are removed prior to the completion of DNA replication; the enzymes that perform this function will be discussed in later chapters.

Figure 1.6. Initiation of transcription. RNA polymerase initiates RNA synthesis within an unwound bubble region of DNA that is formed by the enzyme at promoters (panel A). The synthetic process begins with the formation of a phosphodiester bond between two ribonucleoside triphosphates, with the initial product consisting of a dinucleotide with triphosphate at the 5′ end (panel B).

As discussed above, the base pairs in a DNA duplex are interior to the molecule, with the two or three hydrogen bonds of the base pairs buried deep in the molecule. Furthermore, the two daughter duplexes after replication have one strand each from the parent molecule and one new strand. It is therefore obvious that the DNA duplex must be separated into its two constituent strands as replication occurs. This process is called DNA unwinding to reflect the loss of the helical turns of the two strands around each other. While some DNA polymerases are capable of driving this DNA unwinding reaction, the replication machinery has a more efficient type of enzyme that carries out this important function, called a “DNA helicase.” DNA helicases unwind the duplex DNA into its two constituent strands while hydrolyzing a ribonucleotide triphosphate (usually ATP) to fuel the reaction (Figure 1.5). While we discuss the enzymes of DNA synthesis separately, this is a good time to point out that the three enzymes introduced so far, DNA polymerases, primases, and helicases, function together and interact physically, influencing the activities of each other in profound ways. Indeed, these and additional proteins involved in DNA replication form an intricate and well-coordinated machine for the duplication of DNA (Chapters 2–4).

During the synthesis of the short Okazaki fragments on the lagging strand as replication proceeds, segments of the lagging-strand template are exposed as single strands following helicase action (Figure 1.5). Single-stranded DNA (ssDNA) is more vulnerable to various forms of damage, and also in a sense more disorganized than duplex DNA, which has a regular helical structure. This leads us to the next important protein in the replication machinery, one that does not have enzymatic function but rather binds to ssDNA and helps protect and organize it. This protein, called ssDNA-binding protein, binds to transient regions of ssDNA as replication proceeds and is critical for the proper functioning of the replication machinery. There is evidence from prokaryotic replication systems that ssDNA-binding protein also plays a key organizational role in “spooling” the ssDNA into a more regular structure. As will be discussed later in the book, ssDNA-binding protein also plays many key roles in DNA repair and other cellular pathways.

As already mentioned, RNA primers become incorporated into the 5′ ends of new DNA strands during replication. These RNA residues must be removed before completion of DNA synthesis, and this job is completed by nucleases of different kinds. To avoid leaving a single-stranded gap at the site of every primer, a DNA polymerase is employed to extend the 3′ end of the preceding Okazaki fragment until it reaches the 5′ end created by nuclease action. Finally, the two adjacent fragments need to be joined together, and this step employs a special enzyme called “DNA ligase” (see Chapters 2–4 for more detailed discussion of Okazaki fragment processing).

As mentioned above, the two parental strands in a DNA duplex are wound around each other once every 10.5 base pairs. Unwinding of these two parental strands during DNA replication has the potential to leave the two daughter duplexes in a hopelessly tangled state, for example with the two daughter duplexes of each human chromosome wrapped around each other millions of times. This potential problem relates to the higher order structure, or topology, of the DNA molecule. To solve this problem, all cells have enzymes, called “DNA topoisomerases” that alter the topology of DNA in various ways. As described in Chapter 7, DNA topoisomerases are critical for completing DNA replication properly, and inhibition of the appropriate topoisomerase does indeed lead to a hopeless tangling of the two daughter duplexes as cells try to divide. DNA topoisomerases are also important during other processes, including transcription. Intriguingly, these enzymes are the targets for a number of chemotherapeutic drugs, including commonly used anticancer and antibacterial agents.

Two additional pathways, described in Chapters 5 and 6, are also needed to successfully complete DNA replication. Because of the inherent directionality of DNA polymerases, the very ends of chromosomes in eukaryotic cells require a special mechanism of replication and a different set of proteins from the normal replication machinery. These chromosome ends also have a special structure, called a “telomere,” and one of the key enzymes in the replication of chromosome ends is a specialized DNA polymerase called “telomerase.” Finally, a special repair pathway called “mismatch repair” increases the fidelity of DNA replication well beyond that which can be achieved with the replication machinery itself; mismatch repair occurs just after DNA replication.