Ординатура / Офтальмология / Английские материалы / Biochemistry of the Eye 2nd edition_Whikehart_2003

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by hydrogen. When a phosphopentose, such as deoxyribose phosphate, is joined to a base, the compound is known as a nucleotide and the four bases, as just given, add the word phosphate to their name, such as, deoxyadenosine 5′-triphosphate (dATP), adenosine 5′-triphosphate (ATP), guanosine 3′,5′-cyclic monophosphate (cGMP; see Chapter 6) and deoxycytidine 5′-monophosphate (dCMP). These are shown in Figure 7–2. Only the first example would be incorporated into a molecule of DNA. The second example either may act as a source of cellular energy (see Chapter 4) or be incorporated into a molecule of RNA. Cyclic GMP, the third example, acts as an intracellular hormone and deoxycytidine 5′-monophosphate, the fourth example, is a breakdown product of DNA.

The 2′-deoxynucleotides of the four bases (see Figure 7–1) are the only nucleotides incorporated into DNA. In order to be incorporated, they must first exist in the triphosphate form. Biochemically, they become bound to DNA by the catalytic activity of a polymerase enzyme. In the process, a diphosphate group is removed from the deoxynucleotide triphosphate. Synthesis proceeds with the addition of the 5′-phosphate end of the added nucleotide to the 3′-end of the growing chain and may be seen in Figure 7–3.

Several features about the DNA chain, as seen in Figure 7–3, should be noted. The DNA, once made, is double-stranded in eukaryotes. That is, in cells having a nucleus. The bases are held together by hydrogen bonds: two bonds between adenine (A) and thymine (T); and three bonds between guanine (G) and cytosine (C). The bases themselves are hydrophobic while the deoxypentose phosphates are hydrophilic. Once formed, the double-stranded DNA (or duplex DNA) forms into a helix with the hydrophobic bases held internally and the deoxypentose phosphates located on the exterior of the helix. Helical DNA is a structure that was predicted by Watson and Crick in 1953. This DNA helix, as shown in Figure 7–4, is known as the B-form. An A-form and a Z-form also occur (Wells et al, 1988). These latter two forms are used by cells in special circumstances. Their structures are beyond the scope of this text, but may be seen in Mathews and van Holde (1990).

Typically, on a strand of duplex DNA, one chain contains the code for the synthesis of specific proteins while the other chain contains

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Figure 7–3

DNA replication by DNA-directed DNA polymerase. The enzyme forms 5′→ 3′ bonds with each incorporated base. The hydrogen bonds A-T and G-C form spontaneously after 5′→ 3′ bonding where the black and white lines are shown. Each triphosphate nucleotide loses a diphosphate group in the catalytic process. The base to be added is determined by the complementary, opposite base on the template strand.

consist of duplex DNA wrapped around the barrel-like structure of histone proteins. Strands of DNA connect each barrel while other nonhistone proteins are bound to the DNA at these strands. Histone proteins assist DNA in compacting itself while nonhistone proteins are involved with the control of DNA and RNA processing (Figure 7–6).

Nucleic Acids • 195

Figure 7–4

A representation of the B-form of the double-helical structure of DNA (duplex DNA). This is its most common form. Note the presence of both a major and a minor groove in the helix. Functional proteins bind to the DNA at its major groove.

DNA REPLICATION

When a cell divides, it reproduces its DNA by a process known as replication. Replication is semiconservative. This means that one original strand of the “parent” duplex DNA becomes incorporated into each of the two new “daughter” duplex DNA molecules. This is to say that one coding strand and one complementary strand of each of the two new strands came from the original DNA before division.

Since the DNA in each chromosome is so long, replication takes place in several locations at one time. It requires many proteins to carry out replication. Some of these proteins, such as DNA polymerase, are enzymes. Each location of active replication is referred to as a replication fork in which the parental DNA is the stem and each daughter DNA is a tine of the fork. Several requirements for successful replication make this process complicated. For example, the helix must be unwound before replication and the synthesis of each strand must proceed from the 5′-end of each added nucleotide to the 3′-end of each growing strand. This may not seem complicated intuitively. However, when one realizes that each duplex DNA has its member strands oriented in opposite 5′→3′ directions, a paradox becomes apparent if replication can only proceed in one direction. The problem is solved by the genetic machinery of the cell (Figure 7–7). The figure shows a

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Figure 7–5

Unraveling a metaphase chromosome (two duplex DNA molecules or chromatids). The supercoiled structures can be untwisted to show chromatin fibers containing nucleosomes (histones and wound DNA) joined by linker DNA. The nonhistone proteins are bound to DNA in between each nucleosome.

Figure 7–6

The appearance of chromatin fibers (Figure 7–5) in greater detail. H1 histones appear to stabilize the DNA on the nucleosome.

diagram of a replication fork with parental DNA on the left. In the top daughter duplex DNA, the new strand is synthesized in the conventional 5′→3′ direction. This strand is known as the leading strand. In the bottom daughter duplex DNA, the new strand is made in discrete, short discontinuous fragments (5′→3′) known as Okazaki fragments, named for Reiji Okazaki, the scientist who discovered them (Ogawa, Okazaki, 1980). Each short fragment in the lagging strand (although made 5′→3′) is successively formed backwards in the same direction as that of the opening fork, that is, in the same direction as the growing, leading strand. The fragments are then joined by a ligase enzyme and

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Figure 7–7

Sequence scheme of daughter DNA on the leading and lagging strands. The numbers on the Okazaki fragments (first through fourth) indicate the order of synthesis of each fragment in time while the arrows indicate their 5′→ 3′ sequence.

Figure 7–8

The overall effective sequence of synthesis of both the leading and lagging strands.

become a continuous strand. In effect, the synthesis of each strand appears as though it was unidirectional (Figure 7–8). The molecular assembly of a replication fork may be seen in Figure 7–9. As can be seen in the figure, several proteins contribute to the replication process. DNA helicase, an enzyme, breaks the hydrogen bonds between the bases to form single strands. Bound to the DNA helicase is a second enzyme: RNA primase. This enzyme periodically synthesizes short strands of primer RNA that bind to one of the separated parental strands. One of the primer strands becomes the template for the new lagging strand and the RNA primer becomes the initiator for that lagging strand. It is important that this parental strand remains a single strand at this stage. For this reason, double-helix-destabilizing proteins temporarily bind to it. The parental template for the lagging strand loops around and passes through a DNA polymerase III enzyme complex that is actually two polymerase enzymes. The lower polymerize III enzyme synthesizes new DNA bound to the RNA primer to form

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Figure 7–9

The actual appearance of a replication fork. White arrows point out the proteins involved in replication. Although similar to Figures 7–7 and 7–8, it may be seen that the parental DNA forming the lagging strand template must first loop back toward the lower DNA polymerase III molecule before an Okazaki fragment can be formed. For this reason, helixdestabilizing proteins prevent premature helical formation.

an Okazaki fragment on the lagging strand, while the other upper polymerase III forms the new DNA chain for the leading strand. A little further out on the lagging strand a complex of DNA polymerase I and ligase hydrolyze the RNA portion of the primer and complete the gaps between the DNA Okazaki fragments to form a continuous strand. The ligase enzyme binds the last nucleotide to join the fragment with the new growing strand.

RIBONUCLEIC ACID

Ribonucleic acid, chemically, is similar to DNA except for two important differences: (1) ribose replaces deoxyribose as a pentose; and (2) uracil (as uridine ribose monophosphate) replaces thymine as a base in most cases. Uridine 5′-monophosphate is shown in Figure 7–10 and its structure should be compared with the bases in Figure 7–1. As a rule, RNA is single-stranded, but there are notable exceptions with regions of both transfer and ribosomal RNA (to be discussed) as well as with some RNA containing viruses.

In eukaryotic cells, the processes of producing proteins require the existence of four different kinds of RNA. These are listed in Table 7–1.

Heterogeneous nuclear RNA (hnRNA) is the initial coding RNA product made from DNA. Messenger RNA (mRNA) is a cellular refinement of hnRNA, made in the nucleus, and represents the form of RNA that carries the exact code for protein sequencing. It is the form used in the process of protein synthesis known as translation. It is interesting that this RNA form comprises only 3% of the total RNA present at a given time. Such a low value indicates the strict control necessary for the rate of protein synthesis. Transfer RNA (tRNA) is a short chain RNA that attaches to specific amino acids and transports them to ribosomes where proteins are synthesized. Ribosomal RNA (rRNA) represents 75% of all