Proteins Required for DNA Replication
Two questions arise in separating the two strands of the original DNA so that it can be replicated. The first is how to achieve continuous unwinding of the double helix. This question is complicated by the fact that prokaryotic DNA exists in a supercoiled, closed-circular form (see “Tertiary Structure of DNA: Supercoiling”). The second related question is how to protect single-stranded stretches of DNA that are exposed to intracellular nucleases as a result of the unwinding.
An enzyme called DNA gyrase (class II topoisomerase) catalyzes the conversion of relaxed, circular DNA with a nick in one strand to the supercoiled form with the nick sealed that is found in normal prokaryotic DNA (Figure 10.8). A slight unwinding of the helix before the nick is sealed introduces the supercoiling. The energy required for the process is supplied by the hydrolysis of ATP. Some evidence exists that DNA gyrase causes a double-strand break in DNA in the process of converting the relaxed, circular form to the supercoiled form.
In replication, the role of the gyrase is somewhat different. The prokaryotic DNA is negatively supercoiled in its natural state; however, opening the helix during replication would introduce positive supercoils ahead of the replication fork. To see this phenomenon for yourself, look for an old phone with a coiled cord and try straightening out a section of the cord. You will be able to see the result in the coils ahead. If the replication fork continued to move, the torsional strain of the positive supercoils would eventually make further replication impossible. DNA gyrase fights these positive supercoils by putting negative supercoils ahead of the replication fork (Figure 10.9). A helix-destabilizing protein, called a helicase, promotes unwinding by binding at the replication fork. A number ofhelicases are known, including the DnaB protein and the rep protein.
Single-stranded regions of DNA are very susceptible to degradation by nucleases.
If left unchecked, this would make it very difficult to complete replication before DNA damage would occur.
Another protein, called the single-strand bindingprotein (SSB), stabilizes the single-stranded regions by binding tightly to theseportions of the molecule. The presence of this DNA-binding protein protects the single-stranded regions from hydrolysis by the nucleases.
One of the great surprises in studies of DNA replication was the discovery that RNA serves as a primer in DNA replication. In retrospect, it is not surprising atall, because RNA can be formed de novo without a primer, even though DNA synthesis requires a primer. This finding lends support to theories of the origin of life in which RNA, rather than DNA, was the original genetic material. The fact that RNA has been shown to have catalytic ability in several cases has added support to that theory. A primer in DNA replication must have a free 3'-hydroxyl to which the growing chain can attach, and both RNA and DNA can provide this group. The primer activity of RNA was first observed in vivo. In some of the original in vitro experiments, DNA was used as a primer because a primer consisting of DNA was expected. Living organisms are, of course, far more complex than isolated molecular systems and, as a result, can be full of surprises for researchers.
It has subsequently been found that a separate enzyme, called primase, is responsible for copying a short stretch of the DNA template strand to produce the RNA primer sequence. The first primase was discovered in E. coli. The enzyme consists of a single polypeptide chain, with a molecular weight of about 60,000. There are 50 to 100 molecules of primase in a typical E. coli cell. The primer and the protein molecules at the replication fork constitute the primosome. The general features of DNA replication, including the use of anRNA primer, appear to be common to all prokaryotes (Figure 10.9).
The synthesis of two new strands of DNA is begun by DNA polymerase III. The newly formed DNA is linked to the 3'-hydroxyl of the RNA primer, and synthesis proceeds from the 5' end to the 3' end on both the leading and the lagging strands. Two molecules of Pol III, one for the leading strand and one for the lagging strand, are physically linked to the primosome. The resulting multiprotein complex is called the replisome. As the replication fork moves, the RNA primer is removed by polymerase I, using its exonuclease activity. The primer is replaced by deoxynucleotides, also by DNA polymerase I, using its polymerase activity. (The removal of the RNA primer and its replacement with the missing portions of the newly formed DNA strand by polymerase I are the repair function we mentioned earlier.) None of the DNA polymerases can seal the nicks that remain; DNA ligase is the enzyme responsible for the final linking of the new strand. Table 10.3 summarizes the main points of DNA replication in prokaryotes.
Besides the DNA polymerases themselves, many other proteins are involved in replication. DNA gyrase induces negative supercoils in the DNA to compensate for the positive supercoils that would form because of strand separation, and helicase induces strand separation. Single-stranded binding proteins protect the single-stranded regions from nucleases.
Primase primes the synthesis of the lagging strand by the formation of primers, and DNA ligase links pieces of newly formed DNA together.
The primer and the proteins at the replication fork are called the primosome.
The entire complex, including the DNA polymerases, is called the replisome.