Cell & Molecular Biology JUL 16, 2016 To replicate DNA and RNA nucleotide chains, new copies are synthesized from existing ones. This copying process always happens in a "forward" direction, from the 5’ to the 3’ end. During the process the double-stranded DNA is separated into two strands and aligned in opposite directions, complicating the matter. Yao and her team utilized X-ray crystallography to reveal how TLP forms a complex with RNA. From that work, they gleaned insight into the complicated mechanism that TLPs use to add nucleotides in the reverse direction. That structural analysis uncovered a two-step process. First, energy-supplying molecules are recruited and second, a nucleotide is added. That second step is also observed in the forward (5' to 3') reaction. But what is unique to the reverse reaction is the recruitment of energy at the start. The enzyme apparently needs this energy to change the direction from forward to reverse. This is opposite from typical replication. While the basis of the reaction is similar in both cases, from an energetic viewpoint, the reactions are very different; the high energy of the added nucleotide is used for its own attachment with DNA/RNA polymerases, in TLPs the high energy of the incoming nucleotide is used for subsequent nucleotide addition. These differences require the Thg enzyme to use a structurally complicated process that probably makes it unsuitable for DNA replication. "By comparing the molecular mechanisms of forward and reverse reactions in more detail, we would like to fully understand the evolutionary context of DNA replication," concludes Yao.Sources: Science Daily via Hokkaido University, Science Advances DNA is a remarkable molecule. One of its most fundamental properties is that it can be split into two halves, each of which then acts as a template for the replacement of the other half. In other words a single DNA molecule can become two identical molecules, reproducing itself - the basis of life! This copying process is called replication. The DNA molecule is a double helix in shape, and each helix is a polynucleotide, i.e a polymer consisting of a number of nucleotides in a row, coiled up to give this shape. The outside edges of each helix consist of alternating deoxyribose and phosphate groups, held together by strong phosphodiester bonds. The middle part of the double helix consists of pairs of nitrogenous bases - adenine + thymine, or cytosine + guanine, clinging together by much weaker hydrogen bonds. These pairs are called complementary, as their molecules are shaped to fit together, and the hydrogen bonding relies on this closeness of fit. The two polynucleotide strands run in opposite directions. This is called antiparallel. In order to start the replication of DNA, the two sides of it must be peeled apart, like a zip opening. This gives a Y- shaped section to the molecule, known as a replication fork. The two alternating sugar-phosphate "backbone" sections remain attached along their length, and each of the nitrogenous bases remains attached to their deoxyribose, but they are exposed, i.e. unpaired with another base.This unzipping or unwinding process is caused by an enzyme DNA helicase, which moves along the DNA strands, breaking the hydrogen bonds between bases, separating the two strands. The next stages occur under the control of the enzyme DNA polymerase. There are actually two or more copies of this enzyme, operating independently on the original two different DNA strands. Each strand forms a template on which the copied half of that strand is built up. This process is called semi-conservative replication because half of each molecule is kept and used as a template for the formation of the other half.
 DNA replication based on a diagram by Madeleine Price Ball One strand is said to run in the 3' to 5' direction, and the other runs in the 5' to 3' direction. This is a reference to the carbon atoms in deoxyribose which have -OH groups which participate in phosphodiester bond formation. There is in fact a (loose, unreacted) hydroxyl (-OH) group at each end of each DNA strand, one at the 3' and one at the 5' position. If a (single strand of) DNA sequence is written on a page it is written in the format 5' ATT....GCA 3'. DNA polymerase only works in one direction, adding nucleotides to the 3' end of the developing polynucleotide chain alongside the original DNA strand it is attached to. Before any cell divides, it must replicate its DNA, so it briefly contains twice the normal amount of DNA. In normal cell division, the nucleus undergoes the process of mitosis - a single division - then the cell divides to give two daughter cells with identical genetic makeup. DNA replication takes place before this - during the S phase (synthesis phase) of the cell cycle. In the production of sex cells, meiosis takes place - 2 divisions - and each cell produced contains half the genetic material. So the biochemistry of DNA ensures that genetic information is passed reliably from cell to cell and from generation to generation. In reality DNA replication involves the opening out of the double helix at several places, so there are several replication forks. Each replication fork is likely to have a partner fork proceeding in the opposite direction, so that a replication bubble is established.
A replication bubble DNA helicase is sometimes shown as a wedge separating the two strands of DNA. In fact it consists of a number of subunits that join together, forming a ring round one strand of DNA, and this acts like a motor, progressing along that DNA strand, powered by ATP. DNA helicase attaches to DNA where an initiator protein opens a section of DNA with a high proportion of A-T pairs (which have only 2 hydrogen bonds, so are easier to separate). Proteins - single stranded binding proteins (ssBPs) - attach to the exposed single strands of DNA, preventing it from re-annealing (re-joining or coiling up with itself) until new nucleotides are brought in to re-form the double strands. A primase enzyme (a type of RNA polymerase) attaches to a region of single-stranded DNA and produces a primer - an RNA copy of a short section of it. This primer functions as an attachment point for DNA polymerase which then brings in the appropriate (DNA) nucleotides one by one, and causes the formation of phosphodiester bonds. In fact each nucleotide is brought in as the triphosphate version (dATP, dTTP, dGTP, dCTP), and removal of 2 phosphate groups (as pyrophosphate) gives energy to power the synthesis of the developing copied strand. This is similar to the action of ATP in powering cell activity. Only one strand - the lead strand - can be rebuilt directly - the DNA polymerase moving continuously along the template polynucleotide strand in the 3' to 5' direction, thus building up a new strand in the 5' to 3' direction. The other strand - the lag strand - which is running in the opposite direction, has to be rebuilt in sections, so several primases and DNA polymerases are usually involved. The individual sections - 100 to 200 nucleotides long in eukaryotes, possibly 10x as long in prokaryotes - are called Okazaki fragments. There are several versions of most of the enzymes involved in DNA synthesis, and minor differences between prokaryotes and eukaryotes. Topoisomerases cut and reform DNA ahead of DNA helicase, preventing DNA from coiling into tight supercoils at the replication fork. Different DNA polymerases pass along the newly formed DNA double helix converting RNA primer sections into DNA. Some polymerases also carry out a 'proofreading' process. The gaps between Okazaki fragments are joined together by the enzyme DNA ligase so that the strand formed is a complementary copy of the original template strand. |
What is it called when the dna strands run in opposite directions?
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