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An AMP group istransferred first to a Lys residue on the enzymeand then to the 5' phosphate in the nick, (c) The3' -OH group then attacks this phosphate and displaces AMP, leading to the formation of a phosphodiester bond to seal the nick. The AMP is derivedfrom NAD+ in the case of E. coli DNA ligase. TheDNA ligases isolated from a number of other prokaryotic and eukaryotic sources use ATP ratherthan NAD+, and release pyrophosphate rather thannicotinamide mononucleotide (NMN) in step (a).DNA polymeraseIII dimerHelicasesTopoisomerase(DNA gyrase)PrimosomeRNA primerLagging strandFigure 24-16 Coupling the synthesis of leadingand lagging strands with a dimeric DNA polymerase III.
The template for the lagging strand islooped tightly so that the direction of synthesis hasthe same orientation for both strands. As polymerization proceeds, the loop grows until the previousOkazaki fragment is encountered. Here, the polymerase synthesizing the lagging strand must dissociate and reinitiate at a new primer and with a newtight loop. This must be coordinated to keep pacewith that part of the polymerase synthesizing theleading strand.830Part IV Information PathwaysReplication in Eukaryotic Cells Is More ComplexThe DNA molecules in eukaryotic cells are considerably larger thanthose in bacteria and are organized into complex nucleoprotein structures (chromatin) (Chapter 23).
The essential features of DNA replication are the same in eukaryotes and prokaryotes. However, some interesting variations on the general principles discussed above promisenew insights into the regulation of replication and its link with thecell cycle.Origins of replication, called autonomously replicating sequences(ARS), have been identified and studied in yeast.
ARS elements spanregions of about 300 base pairs and contain several conserved sequences that are essential for ARS function. There are about 400 ARSelements in yeast, with most chromosomes having several. Proteinsthat specifically bind the ARS region have been identified in yeast,although their functions are not yet understood.The rate of replication fork movement in eukaryotes (—50 nucleotides/s) is only one-tenth that observed in E. coli. At this rate, replication of an average human chromosome proceeding from a single originwould take more than 500 hours. Instead, replication of human chromosomes proceeds bidirectionally from multiple origins spaced 30,000to 300,000 base pairs apart.
With the exception of the ARS elements ofyeast, the structure of the origins of replication in eukaryotes is notknown. Because eukaryotic chromosomes are almost uniformly muchlarger than bacterial chromosomes, the presence of multiple origins ona eukaryotic chromosome is probably a general rule.As in bacteria, there are several types of DNA polymerases in eukaryotic cells.
Some have been linked to special functions such as thereplication of the DNA in mitochondria. The replication of nuclearchromosomes involves an enzyme called DNA polymerase a, in association with another polymerase called DNA polymerase 5. DNA polymerase a is typically a four-subunit enzyme with similar structureand properties in all eukaryotic cells. One of the subunits has a primase activity. The largest subunit (Mr ~ 180,000) contains the polymerization activity.
DNA polymerase 8 has two subunits. This enzymeexhibits a very interesting association with and stimulation by a protein called proliferating cell rcuclear antigen (PCNA; Mr 29,000) foundin large amounts in the nuclei of proliferating cells. The PCNA fromyeast will function with DNA polymerase 8 from calf thymus, and thecalf thymus PCNA with yeast DNA polymerase <5, suggesting a conservation of the structure and function of these key components of the celldivision apparatus in all eukaryotic cells.
PCNA appears to have afunction analogous to the /3 subunit of E. coli DNA polymerase HI (seeFig. 24-7), forming a circular clamp that greatly enhances the processivity of DNA polymerase 8.DNA polymerase 8, which has a 3 ' ^ 5 ' proofreading exonucleaseactivity, appears to carry out leading strand synthesis. DNA polymerase a has a relatively low processivity, and with its associated primaseit may carry out lagging strand synthesis as part of a eukaryotic replisome.
Another polymerase called DNA polymerase e, may replaceDNA polymerase 8 in some situations, such as in DNA repair.Two other protein complexes, called RFA and RFC (RF stands forreplication factor), have been implicated in eukaryotic DNA replication. Both have been found in organisms ranging from yeast to mammals. RFA is a eukaryotic single-stranded DNA-binding protein, witha function equivalent to the E. coli SSB protein. RFC appears to facilitate the assembly of active replication complexes.SummaryThe integrity of the structure and nucleotide sequence of DNA is of utmost importance to the cell.This is reflected in the complexity and redundancyof the enzyme systems that participate in DNAreplication, repair, and recombination.Replication of DNA occurs with very high fidelity and within a designated time period in the cellcycle.
Replication is semiconservative, with eachstrand acting as a template for a new daughterstrand. The reaction starts at a sequence in theDNA called the origin, and usually proceeds bidirectionally from that point. DNA is synthesizedin the 5'—>3' direction by DNA polymerases. At thereplication fork, the leading strand is synthesizedcontinuously and in the same direction as replication fork movement. The lagging strand is synthesized discontinuously. The fidelity of DNA replication is maintained by (1) base selection by thepolymerase, (2) a 3'—»5' proofreading exonucleaseactivity that is part of most DNA polymerases, and(3) a specific repair system that repairs any mismatches left behind after replication.Most cells have several DNA polymerases. InE.
coli, DNA polymerase III is the primary replication enzyme. DNA polymerase I is responsible forspecial functions during replication, recombination, and repair. DNA polymerase II has a specialized replication activity that allows it to replicatepast DNA lesions in error-prone DNA repair. Replication of the E. coli chromosome involves manyenzymes and protein factors organized into complexes.
Initiation of replication requires binding ofDnaA protein to the origin, strand separation, andthe entry of the DnaB and DnaC proteins to set uptwo replication forks. The action of DnaA is associated with the E. coli membrane and is regulated bythe action of acidic phospholipids. Initiation is theonly phase of replication that is regulated. The process of elongation has different requirements foreach strand. DNA strands are separated by helicases, and the resulting topological strain is relieved by topoisomerases.
Single-strand DNA binding proteins stabilize the separated strands. Insynthesis of the lagging strand, the primosomeprotein complex moves with the fork and regulatesthe synthesis of RNA primers by primase. Synthesis of the leading and lagging strands by DNA polymerase III may be coupled. RNA primers areremoved and replaced with DNA by DNA polymerase I, and nicks are sealed by DNA ligase.A similar pattern of replication occurs in eukaryotic cells, but eukaryotic chromosomes havemultiple replication origins. Several eukaryoticDNA polymerases have been identified.Every cell also has multiple and sometimes redundant systems for DNA repair.
Mismatch repairin E. coli is directed by transient undermethylationof (5')GATC sequences on the newly synthesizedstrand after replication. Other systems recognizeand repair damage caused by environmentalagents such as radiation and alkylating agents,and damage caused by spontaneous reactions ofnucleotides.
Some repair systems recognize andexcise only damaged or incorrect bases (e.g., uracil), leaving an AP (apurinic or apyrimidinic) sitein the DNA. This is repaired by excising and replacing the segment of DNA containing the APsite. Other excision repair systems recognize andremove pyrimidine dimers and other modified nucleotides. Some types of DNA damage can also berepaired by direct reversal of the reaction causingthe damage: pyrimidine dimers are directly converted to monomeric pyrimidines by photolyase,and the methyl group in O6-methylguanine is removed by a specific methyltransferase. Errorprone repair is a specialized and mutagenic replication process observed when DNA damage is soheavy that the need for some replication outweighsthe need to avoid errors.DNA sequences are rearranged in recombination reactions.
Homologous genetic recombinationoccurs between any two DNAs that share sequencehomology. This reaction takes place in meiosis (ineukaryotes) and is one of the processes that createsgenetic diversity. Homologous recombination alsois needed for repair of some types of DNA damage.A Holliday intermediate in which a crossover hasoccurred between the strands of two homologousDNAs is formed during the process. In E. coli, theRecA protein promotes formation of Holliday intermediates and branch migration to extend heteroduplex DNA.Site-specific recombination occurs only at specific target sequences and can also involve a Holliday intermediate.
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