Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies. Kenneth N Kreuzer

      Figure 1.3.The two major base pairs in DNA. The hydrogen bonds within the guanine-cytosine and adenine-thymine base pairs are shown as dotted lines. The N-glycosidic bonds that connect each base to its respective deoxyribose sugar are indicated by the solid line ending in a squiggle. (Hydrogens on the carbon atoms in the bases are not shown.)

      Many DNA repair reactions rely on the information redundancy in the DNA duplex. Damaged or incorrect bases on one of the two strands of DNA can be corrected by using the template information in the other strand to replace the derelict bases. This allows remarkable stability of the DNA sequence in the face of myriad forms of DNA damage, induced by physical agents such as UV and X-rays and the countless chemicals that can damage DNA in diverse ways.

      The information redundancy in DNA is a bit like having a complete backup copy of all the information on your computer, except that the backup is not a silent library that is only accessed in times of need. The backup is built right into the structure of the DNA molecule — neither strand has a primary or secondary information role, rather both strands have equivalent importance. For example, along the length of any particular chromosome, the “template” strand that is used to direct the synthesis of messenger RNA for protein synthesis differs for different proteins.

      The double-helical nature of the DNA molecule has several dramatic implications for the logic of DNA replication. First, the fact that the two strands are aligned in opposite directions means that the mechanisms to replicate the two strands need to be somewhat distinct, assuming replication proceeds in an orderly direction. The only alternative would be to completely unwind the entire molecule and start replication with two single strands — this strategy is not employed in cells, although we will see that it forms the basis of the powerful method of polymerase chain reaction (PCR; see Section 15.2). Second, the winding of the two strands around each other every 10.5 base pairs introduces an incredible topological problem that must be solved for successful replication and cell division. For example, the two strands of the entire content of human DNA in one cell are wound around each other over 600 million times, and yet the two daughter molecules must be completely disentangled from each other for cell division to be successful (i.e., the two daughter cells each receive a full complement of the genome). Third, in a related issue, the winding of the strands implies that something has to spin rather quickly during the process of unwinding the DNA for replication. Considering the bacterial replication process, with its rate of 1000 base pairs replicated per second, either the DNA or the proteins engaged in unwinding/replication need to crank up a spin rate of nearly 6000 rpm ((1000 bp/second × (1 revolution/10.5 bp)) × 60 seconds/minute), faster than the turbine of some jet engines. Adding to the complexity is the fact that multiple replication machineries act on different regions of the same DNA molecule simultaneously.

      1.3The key functions needed for the process of DNA replication

      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

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