Читать книгу Astrobiology - Charles S. Cockell - Страница 116

One of the most crucial events in the cell is DNA replication during cell division. This event underpins what many consider to be at least one characteristic of life – reproduction. DNA replication proceeds in three coordinated steps: initiation, elongation, and termination. This process is worth examining because it illustrates that cells today are complex machines. The enzymes involved in DNA replication amount to a highly coordinated process that cannot have all come together at once. Questions that immediately arise from looking at DNA replication are: What is the minimum molecular requirement for replicating a strand of DNA? When did the parts of the DNA replication apparatus evolve? How did the earliest cells on Earth replicate their DNA? The answers to these questions are not precisely known, but it is instructive to investigate how DNA replication proceeds to understand what is involved in this process in present-day biology.

In a cell, DNA replication begins at specific locations on the DNA, or origins of replication. In bacteria, there is a single origin of replication on their circular genome or chromosome, whereas in eukaryotes, that have longer linear chromosomes, replication is initiated at multiple origins. The unwinding of DNA at these locations and the synthesis of new strands result in a replication fork (Figure 5.14). Let's examine this process in more detail.

Figure 5.14 The replication of DNA. The figure shows some of the diversity of machinery involved in the process.

An enzyme called a helicase is used to separate the two strands of DNA, essentially “unzipping” the hydrogen bonds. As the helicase separates the DNA at the replication fork, the DNA ahead of it is forced to rotate. This process results in a build-up of twists in the DNA, and a resistance becomes established, which, if not dealt with, would eventually halt the progress of the replication fork. A topoisomerase is an enzyme that temporarily breaks the strands of DNA, relieving the tension caused by unwinding the two strands of the DNA helix.

The replication fork generates two single strands of DNA. Each of these strands (Figure 5.14) can then be used as a template to make the corresponding strand, resulting in two new double helices. This process is called semi-conservative replication because only one strand of the parent double helix is conserved in each new DNA molecule that is made.

The first important point to understand about this process of making two new double-stranded DNA molecules is that the process has directionality. The energy for making a new DNA strand is acquired by cleaving the 5′-triphosphate of the nucleotide that is added to the growing DNA chain. This means that DNA synthesis can only proceed from the 5′ to 3′ direction. On one of the strands, this means that the new complementary strand can be made by synthesizing DNA by following the replication fork as it moves forwards. This is called the “leading” strand. The synthesis of the new strand is accomplished by DNA polymerase. The DNA polymerase is an ancient multimeric enzyme that is responsible for assembling the nucleotides into the newly forming DNA strand.

However, on the other strand of DNA, the “lagging” strand, synthesis must always restart near the replication fork as the DNA unzips (Figure 5.14) and move back in the opposite direction to the replication fork because of the directionality of DNA synthesis. This results in the complication that the lagging strand must be synthesized in fragments. To accomplish this task, a small piece of RNA, an RNA primer, is synthesized and attached to the DNA by a primase enzyme (the primase enzyme is a type of RNA polymerase because it makes RNA). DNA synthesis then proceeds from the RNA primer, accomplished by DNA polymerase. The strands of DNA produced in this way are called “Okazaki” fragments. These fragments are finally stitched together with an enzyme called DNA ligase, generating the complete double strand.

There is one other problem during replication. Single-stranded DNA, produced after the helicase has separated the two DNA strands, tends to fold back on itself forming secondary structures. These structures would interfere with the movement of DNA polymerase along the strand. To prevent this, single-strand binding proteins bind to the DNA until the second strand is synthesized, preventing secondary structure formation.

Termination of DNA replication requires that the progress of the DNA replication fork be stopped. Termination involves the interaction between two components: a specific “termination site sequence” in the DNA, and a protein which binds to this sequence to physically stop DNA replication. In various bacterial species, this protein is named the DNA replication terminus site-binding protein, or Ter protein.

Because bacteria have circular chromosomes, termination of replication occurs when the two replication forks that started out from the origin meet each other on the opposite side of the chromosome.