ZOOHCC - 501: Molecular Biology (Theory)
Unit 2: DNA Replication
Molecular Mechanism of Bacterial DNA Replication
A typical bacterial cell has approximately 1-4 million base pairs of DNA, compared to the 3 billion base pairs of the common house mouse (Mus musculus) genome. However, even in bacteria with small genomes, DNA replication involves an incredibly sophisticated and highly coordinated series of molecular events. These events are divided into four main stages: initiation, completion, primer synthesis, and elongation.
Initiation and Unwinding
During initiation, a so-called initiator protein binds to an origin of replication, a base-paired sequence of nucleotides known as oriC. This binding triggers an event that unwinds the DNA double helix into two single-stranded DNA molecules. Several protein groups are involved in this process (Figure 1). For example, DNA helicases serve to break the hydrogen bonds that bind complementary nucleotide bases. These hydrogen bonds are key features of James Watson and Francis Crick's three-dimensional DNA models. As newly unwound chains tend to recombine, another group of proteins called chain binding proteins keep them stable until elongation begins. A third family of proteins, topoisomerases, relieves some of the torsional stress caused by double helix unwinding.
As mentioned earlier, the place where a strand of DNA begins to wrap around two separate single strands is called the origin of replication. when the double helix is unwound, replication proceeds along his two single strands simultaneously, but in opposite directions (i.e., left to right on one strand). , from right to left on the other strand). This creates two replication forks that replicate as they move along the DNA.
Primer Synthesis
Primer synthesis marks the initiation of the actual synthesis of new DNA molecules. Primers are short nucleotides (about 10-12 bases in length) synthesized by an RNA polymerase enzyme called primase. A primer is required because it is the enzyme that actually adds nucleotides to new DNA strands. DNA polymerases can only add deoxyribonucleotides to the 3'-OH groups of existing strands, making synthesis de novo. because it cannot start. Primase, on the other hand, can add ribonucleotides de novo. Then, after extension is complete, the primer is removed and replaced with DNA nucleotides.
Elongation
Finally, elongation (adding nucleotides to the new DNA strand) begins after the primer is added. Synthesis of the growing strand involves adding nucleotides one at a time in the exact order dictated by the original (template) strand. One of the key features of the Watson-Crick DNA model is that adenine is always paired with thymine and cytosine is always paired with guanine. For example, if the original strand is A-G-C-T, the new strand is T-C-G-A.
DNA is always synthesized in the 5' to 3' direction. That is, nucleotides are added only to the 3' end of the growing strand. As shown in Figure 2, the 5' phosphate group of the new nucleotide bonds to the 3' OH group of the last nucleotide of the growing strand. Scientists have yet to identify a polymerase that can add bases to the 5' ends of DNA strands.
DNA polymerase only moves in one direction
After the primer is synthesized on the DNA strand and the DNA strand is unwound, synthesis and extension proceed in only one direction. As mentioned above, the DNA polymerase can only add to her 3′ end, so her 5′ end of the primer remains unchanged. As a result, synthesis is directly carried out only along the so-called leading strand. This instantaneous replication is called continuous replication. The other strand (5′ to her from the primer) is called the lagging strand, and replication along it is called discontinuous replication. The duplex must relax to some extent before initiating synthesis of another primer further up the lagging strand. Synthesis can then proceed from her 3' end of this new primer. Then the double helix unwinds some more, followed by another surge of replication. As a result, replication along the lagging strand only occurs in short, discontinuous bursts
The newly synthesized fragments of DNA along the trailing strand are called Okazaki fragments, named after their discoverer, the Japanese molecular biologist Reiji Okazaki. Okazaki and his colleagues made their findings by performing so-called pulse-chase experiments, in which replicating DNA was exposed to short 'pulses' of isotope-labeled nucleotides and the length of time the cells were exposed to unlabeled nucleotides was varied. I did. This later stage is called ``chasing'' (Okazaki et al., 1968). The labeled nucleotide was incorporated into the growing DNA molecule only during the first few seconds of the pulse. Afterwards, only unlabeled nucleotides were incorporated during the chase. The scientist then centrifuged the newly synthesized DNA and observed that the shorter the trace, the more radioactivity appeared in the "slow" DNA. Sedimentation velocity was determined by size. Small fragments settled more slowly than large ones due to their lighter weight. When the researchers increased the length of the chase, the radioactivity of the 'fast' DNA increased, with little or no increase in the radioactivity of the slow DNA. The researchers correctly interpreted these observations to mean that the short chase times meant that only very small DNA fragments were synthesized along the lagging strand. As the chase got longer and the DNA had more time to replicate, the pieces of the lagging strand began to integrate into longer, heavier and faster sedimenting DNA strands. Scientists now know that Okazaki fragments in bacterial DNA are typically 1,000 to 2,000 nucleotides long, whereas in eukaryotic cells they are only about 100 to 200 nucleotides long.
Termination
DNA replication termination occurs when two replication forks meet on the same stretch of DNA, and the following events occur, but not necessarily in that order: The forks converge until all the DNA in between has been unwound. Fill the remaining gap and ligate. Catenanes are removed. Replication proteins are unloaded.
Summary
The study of DNA replication began shortly after the structure of DNA was elucidated and continues to this day. Although the stages of initiation, unwinding, primer synthesis, and elongation are now understood in their simplest terms, many questions remain unsolved, especially when it comes to replication of eukaryotic genomes. Scientists have been dedicated to the study of replication for decades, and researchers such as Kornberg and Okazaki have made many important breakthroughs. Yet there is still much to learn about replication, including how errors in this process contribute to human disease.
