- The ability of a cell to survive and proliferate in a chaotic environment depends on the accurate duplication of the vast quantity of genetic information carried in its DNA. This duplication process, called DNA replication, must occur before a cell can divide to produce two genetically identical daughter cells.
- Maintaining order in a cell also requires the continual surveillance and repair of its genetic information, as DNA is subjected to unavoidable damage by chemicals and radiation in the environment and by reactive molecules that are generated inside the cell.
- Transmission of chromosomal DNA from one generation to next is crucial for cell propagation. This can only be achieved when chromosomal DNA is accurately replicated, providing two copies of entire genome for faithful distribution into each daughter cell.
- Differences in DNA can produce the variations that underlie the differences between individuals of the same species—or, over time, the differences between one species and another.
- The long-term storage of information without alteration is so important to a cell, however, that even very slow reactions that alter DNA structure can be physiologically significant. Processes such as carcinogenesis and aging may be intimately linked to slowly accumulating, irreversible alterations of DNA.
- Other, nondestructive alterations also occur and are essential to function, such as the strand separation that must precede DNA replication or transcription. In addition to providing insights into physiological processes, our understanding of nucleic acid chemistry has given us a powerful array of technologies that have applications in molecular biology, medicine, and forensic science.
NATURE OF REPLICATION
It is crucial that genetic material reproduced accurately. If the two strands of parental DNA double helix are separated and each of parental strands then act as template to synthesis complementary daughter strand. This model of replication, in which a parental duplex DNA give rise to two identical daughter duplexes DNA, each containing one original parental strand and one new strand, called semi conservative replication.
The alternative method of DNA replication is conservative and dispersive. In conservative replication, the original parental DNA double helix act as a template for new one, one daughter DNA double helix would consist of the original parental DNA, and the other daughter would be totally new DNA double helix. In dispersive replication some parts of the original parental DNA double helix are conserved, and some parts are not. In this method, the parental double helix is broken into double stranded DNA segments.
Meselson and Stahl experiment
Meselson and Stahl experimentally demonstrated the semi conservative replication of DNA in E-coli in 1958. They grew E-coli cells in a medium in which the sole nitrogen source was heavy isotope nitrogen in the form of ammonium chloride. The heavy isotope of nitrogen containing E-coli was then transfer to normal nitrogen medium and allow continuing growing. Sample were harvested at regular intervals the DNA was extracted and its buoyant density determined by centrifugation in cscl density gradient. The isolated DNA showed a single band in the density gradient, midway between the light nitrogen containing DNA bands and heavy nitrogen containing DNA bands. After two generation in light isotope of nitrogen containing medium the isolated DNA exhibited two bands, one with density equal to light DNA other with density equal to that of the hybrid DNA observed after one generation. The result were same those as expected from the semi conservative replication hypothesis.
PROCESS OF DNA REPLICATION
DNA replication is complex process needs lots of enzymes that all works together for the process to takes place. It includes following steps.
- The DNA double helix is normally very stable: the two DNA strands are locked together firmly by the large numbers of hydrogen bonds between the bases on both strands.
- The process of DNA replication begins at origin of replication (ori) by initiator proteins
- A-T base pair is held together by fewer hydrogen bonds than is a G-C base pair. Therefore, DNA rich in A-T base pairs is relatively easy to pull apart, and A-T-rich stretches of DNA are typically found at replication origins.
- The single replicator required for E. coli chromosomal replication is called oriC. Two repeated motifs are critical for oriC function. The 9-mer motif is the binding site for the E. coli initiator, DnaA, and is repeated five times at oriC. The 13-mer motif, repeated three times, is the initial site of ssDNA formation during initiation.
- After the initiator (DnaA) has bound to oriC and unwound the 13-mer DNA, the combination of ssDNA and DnaA recruits a complex of two proteins: the DNA helicase (DnaB) and helicase loader (DnaC).
- DNA helicases, catalyze the separation of the two strands of duplex DNA. These enzymes bind to and move directionally along ssDNA using the energy of nucleoside triphosphate (usually ATP) binding and hydrolysis to displace any DNA strand that is annealed to the bound ssDNA
- To stabilize the separated strands, ssDNA-binding proteins (SSBs) rapidly bind to the separated strands. Binding of one SSB promotes the binding of another SSB to the immediately adjacent ssDNA.
- This is called cooperative binding and occurs because SSB molecules bound to immediately adjacent regions of ssDNA also bind to each other. This SSB–SSB interaction strongly stabilizes SSB binding to ssDNA and makes sites already occupied by one or more SSB molecules preferred SSB-binding sites.
- The supercoils introduced by the action of the DNA helicase are removed by topoisomerases that act on the unreplicated dsDNA in front of the replication fork. These enzymes do this by breaking either one or both strands of the DNA without letting go of the DNA and passing the same number of DNA strands through the break.
- DNA helicase breaks only the hydrogen bonds that hold the two strands of DNA together without breaking any covalent bonds. Although topoisomerases break one or two of the targeted DNA’s covalent bonds, each bond broken is precisely re-formed before the topoisomerase releases the DNA.
- All DNA polymerases require a primer with a free 30 -OH. They cannot initiate a new DNA strand de novo. How, then, are new strands of DNA synthesis started? To accomplish this, the cell takes advantage of the ability of RNA polymerases to do what DNA polymerases cannot: start new RNA chains de novo. Primase is a specialized RNA polymerase dedicated to making short RNA primers (5–10 nucleotides long) on an ssDNA template. These primers are subsequently extended by DNA polymerase. Although DNA polymerases incorporate only deoxyribonucleotides into DNA, they can initiate synthesis using either an RNA primer or a DNA primer annealed to the DNA template
- RNA:DNA hybrid is recognized as a primer template junction by the sliding DNA clamp loader, a sliding clamp is assembled at this site, and a second DNA Pol III enzyme initiates lagging-strand synthesis.
- The 5ʹ-to-3ʹ direction of the DNA polymerization reaction poses a problem at the replication fork, at each replication fork, one new DNA strand is being made on a template that runs in one direction (3ʹ to 5ʹ), whereas the other new strand is being made on a template that runs in the opposite direction (5ʹ to 3ʹ).
- The DNA strand that appears to grow in the incorrect 3ʹ-to-5ʹ direction is actually made discontinuously, in successive, separate, small pieces—with the DNA polymerase moving backward with respect to the direction of replication-fork movement so that each new DNA fragment can be polymerized in the 5ʹ-to-3ʹ direction. The resulting small DNA pieces—called Okazaki fragments after the biochemists who discovered them—are later joined together to form a continuous new strand. The DNA strand that is made discontinuously in this way is called the lagging strand, because the backstitching imparts a slight delay to its synthesis; the other strand, which is synthesized continuously, is called the leading strand.
- The combination of all of the proteins that function at the replication fork is referred to as the replisome.
- For the leading strand, an RNA primer is needed only to start replication at a replication origin; once a replication fork has been established, the DNA polymerase is continuously presented with a base-paired 3ʹ end as it tracks along the template strand. But on the lagging strand, where DNA synthesis is discontinuous, new primers are needed to keep polymerization going.
- The gap, a break remains in the sugar–phosphate backbone of the Okazaki fragments during replication of the lagging DNA strand this nick in the helix is sealed by DNA ligase.
- Replication of genome terminate at terminus region containing multiple copies of 23 base pair ter (for terminus) sequence each acting as recognition site for sequence specific DNA binding protein called tus (terminal utilizing substances) protein.
- When bound to terminal sequence tus protein allow to pass if the fork is moving in one direction, but block the progression if moving in opposite direction. Here it blocks the passage of DNA B helicase, which is responsible for progression of replication fork.
When the DNA double helix was discovered, the feature that most excited biologists was the complementary relationship between the bases on its intertwined polynucleotide chains. It seemed unimaginable that such a complementary structure would not be used as the basis for DNA replication, but as soon as the self-complementary nature of DNA became known, the idea that protein templates might play a role in DNA replication was discarded. It was immensely simpler to postulate that each of the two strands of every parental DNA molecule served as a template for the formation of a complementary daughter strand. Although from the start this hypothesis seemed too good not to be true, experimental support nevertheless had to be generated. Happily, within 5 years of the discovery of the double helix, decisive evidence emerged for the separation of the complementary strands during DNA replication, and enzymological studies showed that DNA alone is the template for the synthesis of new DNA strands.
With these results, the problem of how genes replicate was in one sense solved. But in another sense, the study of DNA replication had only begun. How does DNA replication begin? How are the intertwined DNA strands separated so that they can act as template? What regulates the extent of replication so that daughter cells neither accumulate nor lose chromosomes? Study of these and other questions has revealed that the replication of even the simplest DNA molecule is a complex, multistep process, involving many more enzymes than was initially anticipated following the discovery of the first DNA polymerizing enzyme.
- Molecular biology of the genes (seventh edition) by Watson, Baker, Bell, Gann, Levine, Losick
- Life sciences fundamental and practices sixth edition, pathfinder publication By Pranav Kumar and Usha Mina
- Essential cell biology (fourth edition) by ALBERTS, BRAY, HOPKIN, JOHNSON, LEWIS, RAFF, ROBERTS, WALTER
:- Article Written By Zahra Madraswala