7.8: DNA Replication

Once proposed, the double-helical structure of DNA immediately suggested a simple mechanism for the accurate replication of the information stored in DNA. Each strand contains all of the information necessary to specify the sequence of the complementary (anti-parallel) 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, since they are held together only by weak H-bonding interactions. Normally, the single strands simply re-associate with one another. To replicate DNA the open region has to be stabilized and the catalytic machinery involved recruited and activated. We will consider how this is done in general terms, in practice this is a complex and highly regulated process involving a number of molecular components.

The first two issues 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 the synthesis of a new polynucleotide strand on their own or de novo: they must add nucleotides onto the end of a pre-existing nucleic acid polymer, they depend on an "RNA primer”. The primer is synthesized by a DNA-dependent, RNA polymerases using a mechanistically similar process. RNA Primer synthesis is based on a complementary DNA sequence. Both DNA and RNA polymerases link the 5’ (phosphate) end of a nucleotide triphosphate molecule to the pre-existing 3’ (hydroxyl) end of a nucleic acid polymer; the polymerization reaction is said to proceed in the 5’ to 3’ direction; nucleotides are added to the 3' end (↓). The molecules involved in DNA replication and RNA synthesis rely on signals within the DNA that are recognized by specific proteins; together these determine where and when nucleic acid replication (synthesis) begins and ends.

After the dsDNA molecule has “opened” locally primase, a specialized DNA-dependent, RNA polymerase, binds to and synthesizes a short RNA primer. Because the two strands of the DNA molecule are anti-parallel one primase complex associates with each of the two now separated DNA strands. The result is one RNA primer on each strand. DNA-dependent, DNA polymerases then replace primase and begins to catalyze the deoxynucleotide-addition reaction. Which nucleotides are added is based on which nucleotides are present in the existing DNA strand.

Nucleotides exist in various forms within the cell, including nucleotide monophosphate (NMP), diphosphate (NDP), and triphosphate (NTP) forms. To make the nucleic acid polymerization reaction thermodynamically favorable, the reaction uses the NTP form of the nucleotide monomers, generated through the reaction: \[5^{\prime}\text {P-NMP-OH3 }{ }_{\text {existing n-length polymer }}^{\prime}+5^{\prime} \text {P-NTP-OH3 }{ }_{\text {incoming }}^{\prime}+\mathrm{H}_2 0 \leftrightarrow 5^{\prime} \text { P-NTP-NMP-OH3 }{ }_{n \text {-length polymer }}^{\prime}+\text { diphosphate. }\nonumber\]

During this reaction the terminal diphosphate of the incoming NTP is released (a thermodynamically favorable reaction) and a 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 polymer end that can react with another NTP. In theory, this process could continue until the newly synthesized strand reaches the end of the DNA molecule. The strand synthesized from the original primer is known as the “leading” strand. 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 two independent complexes, in both directions along a DNA molecule. Because the polymerization reaction only proceeds by 3’ addition, as new single stranded regions are opened (→) new primers must be created by RNA primase and then extended by DNA polymerase; these are known as the lagging strands. While there are two leading strands leaving a particular DNA replication start site, there are a number of lagging strands involved.

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. But RNA regions, derived from the primers, are not found in “mature” DNA molecules, so there must be a mechanism that removes them. As it turns out, the DNA polymerase complex, like a number of enzyme systems, contains more than one catalytic activity (analogous to the ATP synthase and pump). When the DNA polymerase complex reaches the upstream nucleic acid chain it runs into an RNA primer region. An RNA exonuclease activity associated with the DNA polymerase complex removes the RNA nucleotides and replaces them with DNA nucleotides. Once the RNA region is removed, a DNA ligase acts to link (generate a covalent phosphodiester bond between) the two DNA molecules. These reactions, driven by nucleotide hydrolysis, end up producing a continuous DNA strand that runs from one end of the chromosome to the other, or in circular chromosomes (found in prokaryotes) all the way around the circle.

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