Molecular Events of DNA Replication (2024)

Scientists have devoted decades of effort to understandinghow deoxyribonucleic acid (DNA) replicates itself. In simple terms, replicationinvolves use of an existing strand of DNA as a template for the synthesis of anew, identical strand. American enzymologist and Nobel Prize winner ArthurKornberg compared this process to a tape recording of instructions for performinga task: "[E]xact copies can be made from it, as from a tape recording, so thatthis information can be used again and elsewhere in time and space" (Kornberg,1960).

In reality, the process of replication is far more complexthan suggested by Kornberg's analogy. Researchers typically utilize simple bacterialcells in their experiments, but they still do not have all the answers,particularly when it comes to eukaryotic replication. Nonetheless, scientistsare familiar with the basic steps in the replication process, and they continueto rely on this information as the basis for continued research andexperimentation.

Primer synthesis marks the beginning of the actual synthesisof the new DNA molecule. Primers are short stretches of nucleotides (about 10to 12 bases in length) synthesized by an RNA polymerase enzyme called primase.Primers are required because DNA polymerases, the enzymes responsible for theactual addition of nucleotides to the new DNA strand, can only adddeoxyribonucleotides to the 3'-OH group of an existing chain and cannot beginsynthesis de novo. Primase, on theother hand, can add ribonucleotides denovo. Later, after elongation is complete, the primer is removed andreplaced with DNA nucleotides.

While studying E. colibacteria, enzymologist Arthur Kornberg discovered that DNA polymerases catalyzeDNA synthesis. Kornberg's experiment involved mixing all of the basic"ingredients" necessary for E. coliDNA synthesis in a test tube, including nucleotides, E. coli extract, and ATP, and then purifying and testing theenzymes involved. Using this method, Kornberg not only discovered DNApolymerases, but he also performed some of the initial work demonstrating howenzymes add new nucleotides to growing DNA chains (Kornberg, 1959).

Scientists have since identified a total of five differentDNA polymerases in E. coli, each witha specialized role. For example, DNA polymerase III does most of the elongationwork, adding nucleotides one by one to the 3' end of the new and growing singlestrand. Other enzymes, including DNA polymerase I and RNase H, are responsiblefor removing the RNA primer after DNA polymerase III has begun its work,replacing it with DNA nucleotides (Ogawa & Okazaki, 1984). When theseenzymes finish, they leave a nick between the section of DNA that was formerlythe primer and the elongated section of DNA. Another enzyme called DNA ligase thenacts to seal the bond between the two adjacent nucleotides.

After a primer is synthesized on a strand of DNA and the DNAstrands unwind, synthesis and elongation can proceed in only one direction. Aspreviously mentioned, DNA polymerase can only add to the 3' end, so the 5' endof the primer remains unaltered. Consequently, synthesis proceeds immediatelyonly along the so-called leading strand. This immediate replication is known ascontinuous replication. The other strand (in the 5' direction from the primer)is called the lagging strand, and replication along it is called discontinuousreplication. The double helix has to unwind a bit before the synthesis ofanother primer can be initiated further up on the lagging strand. Synthesis canthen occur from the 3' end of that new primer. Next, the double helix unwinds abit more, and another spurt of replication proceeds. As a result, replicationalong the lagging strand can only proceed in short, discontinuous spurts(Figure 3).

The fragments of newly synthesized DNA along the laggingstrand are called Okazaki fragments, named in honor of their discoverer,Japanese molecular biologist Reiji Okazaki. Okazaki and his colleagues madetheir discovery by conducting what is known as a pulse-chase experiment, whichinvolved exposing replicating DNA to a short "pulse" of isotope-labeled nucleotidesand then varying the length of time that the cells would be exposed tononlabeled nucleotides. This later period is called the "chase" (Okazaki et al., 1968). The labeled nucleotideswere incorporated into growing DNA molecules only during the initial fewseconds of the pulse; thereafter, only nonlabeled nucleotides were incorporatedduring the chase. The scientists then centrifuged the newly synthesized DNA andobserved that the shorter chases resulted in most of the radioactivityappearing in "slow" DNA. The sedimentation rate was determined by size: smallerfragments precipitated more slowly than larger fragments because of theirlighter weight. As the investigators increased the length of the chases,radioactivity in the "fast" DNA increased with little or no increase ofradioactivity in the slow DNA. The researchers correctly interpreted theseobservations to mean that, with short chases, only very small fragments of DNAwere being synthesized along the lagging strand. As the chases increased inlength, giving DNA more time to replicate, the lagging strand fragments startedintegrating into longer, heavier, more rapidly sedimenting DNA strands. Today,scientists know that the Okazaki fragments of bacterial DNA are typically between1,000 and 2,000 nucleotides long, whereas in eukaryotic cells, they are onlyabout 100 to 200 nucleotides long.

Bacterial and eukaryotic cells share many of the same basicfeatures of replication; for instance, initiation requires a primer, elongationis always in the 5'-to-3' direction, and replication is always continuous alongthe leading strand and discontinuous along the lagging strand. But there arealso important differences between bacterial and eukaryotic replication, someof which biologists are still actively researching in an effort to betterunderstand the molecular details. One difference is that eukaryotic replicationis characterized by many replication origins (often thousands), not just one,and the sequences of the replication origins vary widely among species. On theother hand, while the replication origins for bacteria, oriC, vary in length(from about 200 to 1,000 base pairs) and sequence, except among closely relatedorganisms, all bacteria nonetheless have just a single replication origin(Mackiewicz et al., 2004).

Eukaryotic replication also utilizes a different set of DNApolymerase enzymes (e.g., DNA polymerase δ and DNA polymerase εinstead of DNA polymerase III). Scientists are still studying the roles of the13 eukaryotic polymerases discovered to date. In addition, in eukaryotes,the DNAtemplate is compacted by the way it winds around proteins called histones.This DNA-histone complex, called a nucleosome, poses a unique challenge bothfor the cell and for scientists investigating the molecular details ofeukaryotic replication. What happens to nucleosomes during DNA replication?Scientists know from electron micrograph studies that nucleosome reassemblyhappens very quickly after replication (the reassembled nucleosomes are visiblein the electron micrograph images), but they still do not know how this happens(Annunziato, 2005).

Also, whereas bacterial chromosomes are circular, eukaryoticchromosomes are linear. During circular DNA replication, the excised primer isreadily replaced by nucleotides, leaving no gap in the newly synthesized DNA. Incontrast, in linear DNA replication, there is always a small gap left at thevery end of the chromosome because of the lack of a 3'-OH group for replacementnucleotides to bind. (As mentioned, DNA synthesis can proceed only in the5'-to-3' direction.) If there were no way to fill this gap, the DNA moleculewould get shorter and shorter with every generation. However, the ends of linearchromosomes—the telomeres—have several properties that prevent this.

DNA replication occurs during the S phase of cell division. In E. coli,this means that the entire genome is replicated in just 40 minutes, at a paceof approximately 1,000 nucleotides per second. In eukaryotes, the pace is muchslower: about 40 nucleotides per second. The coordination of the proteincomplexes required for the steps of replication and the speed at whichreplication must occur in order for cells to divide are impressive, especiallyconsidering that enzymes are also proofreading, which leaves very few errors behind.