Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 63
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In the cell, it exists as alarge complex (molecular mass of about 1 × 106 daltons) composed of two Pol III protein subunits plus at leastseven other proteins. Although each cell has only 10–20 copies of the Pol III complex, it is responsible not only forthe elongation of DNA chains but also for the initiation of the replication fork at origins of replication and theaddition of deoxynucleotides to the RNA primers. Polymerase I plays an essential, but secondary, role inreplication that will be described in a later section.
Eukaryotic cells also contain several DNA polymerases. Theenzyme responsible for the replication of chromosomal DNA is called polymerase α. Mitochondria have their ownDNA polymerase, polymerase γ, which replicates the mitochondrial DNA.In addition to their ability to polymerize nucleotides, most DNA polymerases are capable of nuclease activities thatbreak phosphodiester bonds in the sugar-phosphate backbones of nucleic acid chains. The many other enzymes thathave nuclease activity, are of two types: (1) exonucleases can remove a nucleotide only from the end of a chain,and (2) endonucleases break bonds within the chains.
DNA polymerases I and III of E. coli have an exonucleaseactivity that acts only at the 3' terminus (a 3'-to-5' exonuclease activity). This exonuclease activity provides a builtin mechanism for correcting rare errors in polymerization. Occasionally, a polymerase adds to the end of thegrowing chain an incorrect nucleotide, which cannot form a proper base pair with the base in the template strand.The presence of an unpaired nucleotide activates the 3'-to-5' exonuclease activity, which cleaves the unpairednucleotide from the 3'-OH end of the growing chain (Figure 5.19). Because it cleaves off an incorrect nucleotideand gives the polymerase another chance to get it right, the 3'-to-5' exonuclease activity of DNA polymerase is alsocalled the proof-Page 193Figure 5.18Addition of nucleotides to the 3'-OH terminus of a growing strand.
The recognition step isshown as the formation of hydrogen bonds between the A and the T. The chemicalreaction is between the 3'-OH group of the 3' end of the growing chain that attacks theinnermost phosphate group of the incoming trinucleotide.reading or editing function. The proof-reading function can ''look back" only one base (the one added last).Nevertheless,The genetic significance of the proofreading function is that it is an error-correcting mechanism that servesto reduce the frequency of mutation resulting from the incorporation of incorrect nucleotides in DNAreplication.Two unexpected features of DNA replication result from functional constraints that are present in all known DNApolymerases. One constraint is that a polymerase can elongate a newly synthesized DNA strand only at its 3' end(Figure 5.20).
Hence the polymerase can move along the template strand only in the 3'-to-5' direction. The secondconstraint is that DNA polymerase is unable to initiate new chains but rather requires a preexisting primer. How theprocess of DNA replication deals with these constraints is described in the next section.Page 194Figure 5.19The 3'-to-5' exonuclease activity of the proofreading function. Thegrowing strand is cleaved to release a nucleotide containing the base G, whichdoes not pair with the base A in the template strand.Figure 5.20The geometry of DNA replication. The new strand (red) is elongated bythe addition of successive nucleotides to the 3' end as the polymerasemoves along the template strand in the 3'-to-5' direction.5.6—Discontinuous ReplicationIn the model of replication suggested by Watson and Crick, which is illustrated in Figure 5.7, both daughter strandswere supposed to be replicated as continuous units.
However, no known DNA molecule replicates in this way.Because DNA polymerase can elongate a newly synthesized DNA strand only at its 3' end, one of the daughterstrands is made in short fragments, which are then joined together. The reason for this mechanism and theproperties of these fragments are described next.Fragments in the Replication ForkAs we have seen, all known DNA polymerases can add nucleotides only to a 3'-OH group. Thus if both daughterstrands grew in the same overall direction, then each growing strand would need a 3'-OH terminus. However, thetwo strands of DNA are antiparallel, so only one of the growing strands can terminate in a free 3'-OH group; theother must terminate in a free 5' end. The solution to this topological problem is that within a single replicationfork, both strands grow in the 5'-to-3' orientation, which requires that they grow in opposite directions along theparental strands.
One strand of the newly made DNA is synthesized continuously (in the lower fork in Figure 5.21).The other strand (in the upper fork in Figure 5.21) is made in small precursor fragments. The precursor fragmentsare also known as Okazaki fragments, after their discoverer. The size of the precursor fragments is from 1000 to2000 base pairs in prokaryotic cells and from 100 to 200 base pairs in eukaryotic cells. Because synthesis of thediscontinuous strand is initiated only at intervals, at least one single-stranded region of the parental strand is alwayspresent on one side of the replication fork (the upper side in Figure 5.21). Single-stranded regions have been seenin high-resolution electron micrographs of replicating DNA molecules, as indicated by the arrows in Figure 5.22.Another implication of discontinuous replication of one strand is that thePage 1953'-OH terminus of the continuously replicating strand is always ahead of the 5'-P terminus of the discontinuousstrand; this is the physical basis of the terms leading strand and lagging strand that are used for the continuouslyand discontinuously replicating strands, respectively.Next, we examine how synthesis of a precursor fragment is initiated.Initiation by an RNA PrimerAs emphasized earlier, DNA polymerases cannot initiate the synthesis of a new strand, so a free 3'-OH is needed.In most organisms, initiation is accomplished by a special type of RNA polymerase.
RNA is usually a singlestranded nucleic acid consisting of four types of nucleotides joined together by 3'-to-5' phosphodiester bonds (thesame chemical bonds as those in DNA). Two chemical differences distinguish RNA from DNA (Figure 5.23). Thefirst difference is in the sugar component.
RNA contains ribose, which is identical to the deoxyribose of DNAexcept for theFigure 5.21Short fragments in the replication fork. For each tract of base pairs, thelagging strand is synthesized later than the leading strand.presence of an -OH group on the 2' carbon atom. The second difference is in one of the four bases: The thyminefound in DNA is replaced by the closely related pyrimidine uracil (U) in RNA. In RNA synthesis, a DNA strand isused as a template to form a complementary strand in which the basesFigure 5.22Electron micrograph (A) and an interpretive drawing (B) of a replicating θ molecule of phage λDNA.
In the electron micrograph, each arrow points to a short, single-stranded region ofunreplicated DNA near the replication fork; the single-stranded regions are somewhat difficult to see, butthey are apparent on close inspection.(Electron micrograph courtesy of Manuel Valenzuela.)Page 196in the DNA are paired with those in the RNA.
Synthesis is catalyzed by an enzyme called an RNA polymerase.RNA polymerases differ from DNA polymerases in that they can initiate the synthesis of RNA chains without aprimer.DNA synthesis is initiated by using a short stretch of RNA that is base-paired with its DNA template. The size ofthe primer differs according to the initiation event. In E. coli, the length is typically from 2 to 5 nucleotides; ineukaryotic cells, it is usually from 5 to 8 nucleotides. This short stretch of RNA provides a primer onto which aDNA polymerase can add deoxynucleotides (Figure 5.24).
The RNA polymerase that produces the primer for DNAsynthesis is called primase. The primase is usually found in a multienzyme complex composed of 15 to 20polypeptide chains and called a primosome. While it is being synthesized, each precursor fragment in the laggingstrand has the structure shown in Figure 5.25.The Joining of Precursor FragmentsThe precursor fragments are ultimately joined to yield a continuous strand of DNA.
This strand contains no RNAsequences, so the final stitching together of the lagging strand requires• Removal of the RNA primer• Replacement with a DNA sequence• Joining where adjacent DNA fragments come into contactIn E. coli, the first two processes are accomplished by DNA polymerase I, and joining is catalyzed by the enzymeDNA ligase, which can link adjacent 3'-OH and 5'-P groups at a nick.
How this is done is shown in Figure 5.26.Pol III extends the growing strand until the RNA of the primer of the previously synthesized precursor fragment isreached. Where the DNA and RNA segments meet, there is a single-strand interruption, or nick. The E. coli DNAligase cannot seal the nick because a triphosphate is present (it can link only a 3'-OH and a 5'-monophosphate).Here, however, DNA polymerase I takes over.
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