Once it was proposed, the double-helical structure of DNA immediately suggested a simple mechanism for the accurate duplication of the information stored in DNA. Each strand contains all of the information necessary to specify the sequence of the complementary strand. The process begins when a dsDNA molecule opens to produce two single-stranded regions. Where DNA is naked, that is, not associated with other molecules (proteins), the opening of the two strands can occur easily. Normally, the single strands simply reassociate with one another. To replicate DNA the open region has to be stabilized and the catalytic machinery organized. We will consider how this is done only in general terms, in practice this is a complex and highly regulated process involving a number of components.
The first two problems we have to address may seem arbitrary, but they turn out to be common (conserved) features of DNA synthesis. The enzymes (DNA-dependent, DNA polymerases) that catalyze the synthesis of new DNA strands cannot start synthesis on their own, they have to add nucleotides to an existing nucleic acid polymer. In contrast, the catalysts that synthesize RNA (DNA-dependent, RNA polymerases) do not require a pre-existing nucleic acid strand, they can start the synthesis of new RNA strand, based on complementary DNA sequence, de novo. Both DNA and RNA polymerases link the 5’ end of a nucleotide triphosphate molecule to the pre-existing 3’ end of a nucleic acid molecule. This polymerization reaction is said to proceed in the 5’ to 3’ direction. As we will see later on, the molecules involved in DNA replication and RNA synthesis rely on signals within the DNA that are recognized by proteins and which determine where synthesis starts and stops, and when nucleic acid replication occurs, but for now let us assume that some process has determined where replication starts. We begin our discussion with DNA replication.
The first step in DNA replication is to locally open up the dsDNA molecule. A specialized RNA-dependent, DNA polymerase , known as primase, collides with, binds to, and synthesizes a short RNA molecule, known as a primer. Because the two strands of the DNA molecule point in opposite directions (they are anti-parallel), one primase complex associates with each DNA strand, and two primers are generated, one on each strand. Once these RNA primers are in place, the DNA-dependent, DNA polymerases replaces the primase and begins to catalyze the nucleotide-addition reaction; which nucleotide is added is determined by which nucleotide is present in the existing DNA strand. The nucleotide addition reaction involves various nucleotides colliding with the DNA-primer-polymerase complex; only the appropriate nucleotide, complementary to the nucleotide residue in the existing DNA strand is bound and used in the reaction.
Nucleotides exist in various phosphorylated forms within the cell, including nucleotide monophosphate (NMP), nucleotide diphosphate (NDP), and nucleotide triphosphate (NTP). To make the nucleic acid polymerization reaction thermodynamically favorable, the reaction uses the NTP form of the nucleotide monomers, generated through the reaction:
(5’P)NTP(3’OH) + (5’P)NTP(3’OH) + H20 ⟷ (5’P)NTP-NMP(3’OH) + diphosphate.
During the reaction the terminal diphosphate of the incoming NTP is released (a thermodynamically favorable reaction) and the nucleotide mono-phosphate is added to the existing polymer through the formation of a phosphodiester [-C-O-P-O-C] bond. This reaction creates a new 3' OH end for the polymer that can, in turn, react with another NTP. In theory, this process can continue until the newly synthesized strand reaches the end of the DNA molecule. For the process to continue, however, the double stranded region of the original DNA will have to open up further, exposing more single-stranded DNA. Keep in mind that this process is moving, through independent complexes, in both directions along the DNA molecule. Because the polymerization reaction only proceeds by 3’ addition, as new single stranded regions are opened new primers must be created (by primase) and then extended (by DNA polymerase). If you try drawing what this looks like, you will realize that i) this process is asymmetric in relation to the start site of replication; ii) the process generates RNA-DNA hybrid molecules; and iii) that eventually an extending DNA polymerase will run into the RNA primer part of an “upstream” molecule. However, there is a complexity: RNA regions are not found in “mature” DNA molecules, so there mush be a mechanisms that removes them. This is due to the fact that the DNA polymerase complex contains more than one catalytic activity. When the DNA polymerase complex reaches the upstream nucleic acid chain it runs into this RNA containing region; an RNA exonuclease activity removes the RNA nucleotides and replaces them with DNA nucleotides using the existing DNA strand as the primer. Once the RNA portion is removed, a DNA ligase activity acts to join the two DNA molecules. These reactions, driven by nucleotide hydrolysis, end up producing a continuous DNA strand. For a dynamic look at the process check out this video208 which is nice, but “flat” (to reduce the complexity of the process) and fails to start at the beginning of the process.
Evolutionary considerations: At this point you might well ask yourself, why (for heavens sake) is the process of DNA replication so complex. Why not use a DNA polymerase that does not need an RNA primer, or any primer for that matter, since RNA polymerase does not need a primer? Why not have polymerases that add nucleotide equally well to either end of a polymer? That such a mechanism is possible is suggested by the presence of enzymes in eukaryotic cells that can carry out the addition of a nucleotide to the 5’ end of an RNA molecule (the 5’ capping reaction associated with mRNA synthesis) that we will briefly considered later on. But, such activities are simply not used in DNA replication. The real answer is that we are not sure of the reasons. These could be evolutionary relics, a process established within the last common ancestor of all organisms and extremely difficult or impossible to change through evolutionary mechanisms, or simply worth the effort (in terms of its effects on reproductive success). Alternatively, there could be strong selective advantages associated with the system that preclude such changes. What is clear is that this is how the system appears to function in all known organisms, so for practical purposes, we have to remember some of the key details involved, these include the direction of polymer synthesis and the need (in the case of DNA) of an RNA primer.
- DNA replication video: http://bcove.me/x3ukmq4x