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24-7.Table 24-1 Comparison of DNA polymerases oiE. coliDNA polymeraseIIIIIIStructural gene*polApolB (dinA)polC (dnaE)Subunits1>4>10Mr103,00088,000f-900,0003'—»5' Exonuclease (proofreading)5'->3' ExonucleasePolymerization rate (nucleotides/s)Processivity (nucleotides added beforedissociation)YesYesYesYesNoNo16-20~7250-10003-200>10,000>500,000xFor enzymes with more than one subunit, the gene listed encodes the subunit with polymerization activity Gene names in parentheses represent earlier designations of the same gene (p 815)The acronym din stands for damage mducible, din genes were originally identified as thosethat were induced as part of the SOS response to heavy DNA damage, as described later inthis chapter" Polymerization subunit only DNA polymerase II shares several subunits with DNA polymeraseIII, including the ft, y, and 8 subunits (see Table 24-2) and possibly othersTable 24-2 Subunits of DNA polymerase III of E.
coliSubunita€0Ty88'XPxMr132,00027,00010,00071,00052,00035,00033,00015,00012,00037,000GeneFunction*1 CorepolC (dnaE) Polymerization activitydnaQ (mutB) 3'—»5' Proofreading exonucleaseJ subunitsholEStable template binding; corednaXenzyme dimerizationEnhanced processivitydnaX*holAEnhanced processivityholBholCholBdnaNRequired for optimal processivityWhere no function is listed, the molecular role of the subunit is not entirely clear." The y subunit is encoded by a portion of the gene for the r subunit, such that the amino-terminal 809c of the r subunit has the same amino acid sequence as the y subunit The y subunit isgenerated by a translational frameshifting mechanism (see Box 26-1) that leads to prematuretranslational termination(a)rte -HZii ^HIBIBSi(b)Figure 24-7 The two fi subunits of E.
coli polymerase III (shown in gray and light blue) form a circular clamp that surrounds DNA, shown in (a)from above as a ribbon structure and in (b) fromthe side as a space-filling model. The clamp slidesalong the DNA, enhancing the processivity of thepolymerase by preventing its dissociation. The yand 8 subunits of DNA polymerase III facilitatethe binding of the p subunits to DNA.*#••^^Figure 24-8 The Klenow fragment of E. coli DNApolymerase I, produced by proteolytic treatment ofthe polymerase, includes the polymerization activityof the enzyme.
The horizontal groove evident onthis face of the protein is the likely binding sitefor DNA.^NickRNAorDNA1I1111I1TemplateDNA strandIDNApolymerase I3151!om1IIiii1i3'i5'dNMPsdNTPsDNA polymerase I is far from irrelevant, however. This enzymeserves a host of "clean-up" functions during replication, recombination,and repair, as discussed later in the chapter. These special functionsare enhanced by an additional enzymatic activity of DNA polymeraseI, a 5'—>3' exonuclease activity. This activity is distinct from the 3'-»5'proofreading exonuclease and is located in a distinct structural domainthat can be separated from the enzyme by mild protease treatment.When the 5'—»3' exonuclease domain is removed, the remaining fragment (MT 68,000) retains the polymerization and proofreading activities, and is called the large or Klenow fragment.
The structure of theKlenow fragment has been determined, and it is this fragment of DNApolymerase I that is depicted in Figure 24-8. The 5'-*3' exonucleaseactivity of intact DNA polymerase I permits it to extend DNA strandseven if the template is already paired to an existing strand of nucleicacid (Fig. 24-9). Using this activity, DNA polymerase I can degrade ordisplace a segment of DNA (or RNA) paired to the template and replace it with newly synthesized DNA. Most other DNA polymerases,including DNA polymerase III, lack a 5'->3' exonuclease activity.DNA Replication Requires Many Enzymesand Protein FactorsNick5'3'iIIIIIIr3"51Figure 24-9 The 5 '->3' exonuclease of DNA polymerase I can remove or degrade an RNA or DNAstrand paired to the template, as the polymeraseactivity simultaneously replaces the degradedstrand.
These activities are important for the roleof DNA polymerase I in DNA repair and in removalof RNA primers during replication, as describedlater in this chapter. The strand of nucleic acid(DNA or RNA) to be removed is shown in green;the replacement strand is shown in red. A nick (aphosphodiester bond broken to leave a free 3' OHand 5' phosphate) is found where DNA synthesisstarts. After synthesis, a nick remains where DNApolymerase I dissociates.
This action of polymeraseI has effectively extended the nontemplate DNAstrand and moved the nick down the DNA, a process that is sometimes called nick translation.824We now know that replication in E. coli requires not just a single DNApolymerase but 20 or more different enzymes and proteins, each performing a specific task. Although not yet obtained as a physical entity,the entire complex has been called the DNA replicase system or thereplisome. The enzymatic complexity of replication reflects the requirements imposed on the process by the structure of DNA.
We willintroduce some of the major classes of replication enzymes by considering the problems that they overcome.To gain access to the DNA strands that are to act as templates thetwo parent strands must be separated. This is generally accomplishedby enzymes called helicases, which move along the DNA and separatethe strands using chemical energy from ATP.
Strand separation creates topological stress in the helical DNA structure, which is relievedby the action of topoisomerases (Chapter 23). The separated strandsare stabilized by DNA-binding proteins. Primers must be present orsynthesized before DNA polymerases can synthesize DNA. The primers are generally short segments of RNA laid down by enzymes calledprimases. Ultimately, the RNA primers must be removed and replaced by DNA.
In E. coli, this is one of the many functions of DNApolymerase I. After removal of the RNA segments and filling in of thegap with DNA, there remain points in the DNA backbone where aphosphodiester bond is broken. These breaks, called nicks, must besealed by enzymes called DNA ligases. All of these processes must becoordinated and regulated. The interplay of these and other enzymeshas been best characterized in the E.
coli system.Chapter 24 DNA Metabolism825Replication of the E. coli Chromosome Proceeds in StagesThe synthesis of a DNA molecule can be divided into three stages:initiation, elongation, and termination. These are distinguished by differences in the reactions taking place and in the enzymes required. Inthe next two chapters we will see that the synthesis of the other majorbiological polymers, RNAs and proteins, can be similarly broken downinto the same three stages, each with unique characteristics.
Theevents described below reflect information derived from in vitro experiments using purified E. coli proteins.Initiation The E. coli replication origin, called oriC, consists of 245base pairs, many of which are highly conserved among bacteria. Thegeneral arrangement of the conserved sequences is illustrated in Figure 24-10. The key sequences for this discussion are two series of shortrepeats; three repeats of a 13 base pair sequence and four repeats of a9 base pair sequence.Tandem array ofthree 13 bp sequencesBinding sites for DnaA protein,four 9 bp sequencesConsensus sequenceGATCTNTTNTTTTConsensus sequenceTTATCCACAAt least eight different enzymes or proteins (summarized in Table24-3) participate in the initiation phase of replication. They open theDNA helix at the origin and establish a prepriming complex that setsthe stage for subsequent reactions.
The key component in the initiationTable 24-3 Proteins required to initiate replication at theE. coli originProteinMrNumber ofsubunitsDnaA protein50,0001DnaB protein(helicase)DnaC protein300,0006*29,000119,0002HUPrimase (DnaG protein)SSBRNA polymeraseDNA topoisomerase II(gyrase)60,00075,600454,000400,000* Subunits in these cases are identical14*64FunctionOpens duplex atspecific sites in originUnwinds DNARequired for DnaB bindingat originHistonelike protein;stimulates initiationSynthesizes RNA primersBinds single-stranded DNAFacilitates DnaA activityRelieves torsional straingenerated by DNAunwindingFigure 24-10 The arrangement of sequences inthe E.
coli replication origin, called oriC The repeated sequences are shaded in color. The term"consensus sequence" is used to describe a repeatedsequence that varies somewhat from one copy tothe next; it depicts the most common nucleotideresidues found at each position in the sequence (Nrepresents any of the four nucleotides). Individualcopies of the repeated sequence may differ from theconsensus at one or several positions.
The arrowsindicate the orientations of the nucleotide sequences.Part IV Information Pathways826Figure 24-11 A model for initiation of replicationat the E. coli origin, oriC. (a) About 20 DnaA protein molecules, each with a bound ATP, bind at thefour 9 base pair repeats. The DNA is wrappedaround this complex, (b) The three 13 base pairrepeats are then denatured sequentially to givethe open complex, (c) The DnaB protein binds tothe open complex, with the aid of DnaC protein,and the DnaB helicase activity further unwindsthe DNA in preparation for priming and DNAsynthesis.Supercoiledtemplateprocess is the DnaA protein (Fig. 24-11). A complex of about 20 DnaAprotein molecules binds to the four 9 base pair repeats in the origin.
Ina reaction that requires ATP and is facilitated by the bacterial histonelike protein HU, the DnaA protein recognizes and successively denatures the DNA in the region of the three 13 base pair repeats, which arerich in A=T pairs. The DnaB protein then binds to this region in areaction that requires the DnaC protein. The DnaB protein is a helicase that unwinds the DNA bidirectionally, creating two potential replication forks. If the E. coli single-strand DNA-binding protein (SSB)and DNA gyrase (DNA topoisomerase II) are added to this reaction invitro, thousands of base pairs are rapidly unwound by the DnaB helicase, proceeding out from the origin.
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