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Telcjmerase

Telcjmerase

6bp RNA

6bp RNA

TELOMERASE RNA RECOGNIZES G bp RF.PEAT

FIGURE 5.29 Telomerase Replaces Repeats at the Ends of Chromosomes

Telomerase RNA recognizes the tandem repeats at the end of linear DNA. The RNA of telomerase sticks out beyond the chromosome ends and serves as a template for addition of new DNA repeats that will repair the segment lost during the last DNA replication.

FIGURE 5.29 Telomerase Replaces Repeats at the Ends of Chromosomes

Telomerase RNA recognizes the tandem repeats at the end of linear DNA. The RNA of telomerase sticks out beyond the chromosome ends and serves as a template for addition of new DNA repeats that will repair the segment lost during the last DNA replication.

each replication cycle, the chromosomes are, in fact, shortened and several of the telomere repeats are lost. However, no unique coding information is lost. Furthermore, in cells where the enzyme telomerase is present, the lost DNA is later replaced by adding several of the six-base-pair units to the 3'-end after each replication cycle (Fig. 5.29).Telomerase carries a small segment of RNA, complementary to the six-base-pair telomere repeat. This allows it to recognize the telomeres and reminds it what sequence to add.

After telomerase has elongated the 3'-ends, the complementary strand can be filled in by normal RNA priming followed by elongation by DNA polymerase and joining by ligase. The telomere repeats also protect the ends of chromosomes against degradation by exonucleases.

Telomere repeat sequences have been remarkably conserved throughout evolution although some variation is seen. The characteristic TTAGGG repeat of vertebrates is also found in the protozoan Trypanosoma, while the sequence in the protozoans Paramecium and Tetrahymena, TTGGGG, differs by only one base. Many insects have the five base repeat, TTAGG, whereas the flowering plant Arabidopsis has a seven base repeat, TTTAGGG. However, recent data indicates that this is not typical of all plants, indeed, several monocotyledonous plants have the same TTAGGG repeat as vertebrates. Among the fungi Aspergillus nidulans has TTAGGG whereas its close relative ligase Enzyme that joins up DNA fragments end to end telomerase Enzyme that adds DNA to the telomere of a eukaryotic chromosome

Eukaryotic Chromosomes Have Multiple Origins 129

FIGURE 5.30 Eukaryotic Chromosome Replication Bubbles

Numerous openings in the DNA, or replication bubbles occur at the sites of replication in eukaryotic chromosomes. The longer replication continues, the larger the bubbles. The bubbles eventually merge together which separates the newly replicated DNA molecules (not shown).

FIGURE 5.31 Eukaryotic DNA Replication

A) The process begins with the binding of a primase which produces an RNA primer. Subsequently, DNA polymerase a binds and initiates a short segment of DNA called initiator DNA (iDNA).

B) Replication factor C associates with the iDNA and is C) helpful in positioning DNA polymerase S. D) DNA polymerase S elongates the new DNA strand.

FIGURE 5.31 Eukaryotic DNA Replication

A) The process begins with the binding of a primase which produces an RNA primer. Subsequently, DNA polymerase a binds and initiates a short segment of DNA called initiator DNA (iDNA).

B) Replication factor C associates with the iDNA and is C) helpful in positioning DNA polymerase S. D) DNA polymerase S elongates the new DNA strand.

Aspergillus oryzae has a double-length repeat—TTAGGGTCAACA. One strange exception to this general pattern is the fruit fly, Drosophila, which has telomeres consisting of tandem sequences generated by successive transposition of two retrotrans-posons (HeT-A and TART) instead of being synthesized by telomerase.

Eukaryotic Chromosomes Have Multiple Origins

Eukaryotic chromosomes are often very long and have numerous replication origins scattered along each chromosome. Replication is bi-directional, as in bacteria. A pair of replication forks starts at each origin of replication and the two forks then move in opposite directions (Fig. 5.30). The bulges where the DNA is in the process of division are often called replication bubbles.

A vast number of replication origins function simultaneously during eukaryotic DNA replication. For example, there are estimated to be between 10,000 and 100,000 replication origins in a dividing human somatic cell. This creates major problems in synchronization. Synthesis at each origin must be coordinated to make sure that each chromosome is completely replicated. Conversely, each origin must initiate once and once only during each replication cycle in order to avoid duplication of DNA segments replication bubble (replication eye) Bulge where DNA is in the process of replication

Eukaryotic Chromosomes Have Multiple Origins 129

FIGURE 5.30 Eukaryotic Chromosome Replication Bubbles

Numerous openings in the DNA, or replication bubbles occur at the sites of replication in eukaryotic chromosomes. The longer replication continues, the larger the bubbles. The bubbles eventually merge together which separates the newly replicated DNA molecules (not shown).

Eukaryotic chromosomes are much longer than bacterial ones and have m ultiple replication origins.

that have already been replicated. This is achieved by a protein complex, known as replication licensing factor (RLF), which binds to the DNA next to each origin before each replication cycle and is displaced during replication. Only when RLF is present is DNA replication permitted.

Synthesis of eukaryotic DNA resembles that in bacteria in most general respects:

a) it is semi-conservative b) one strand is made in short fragments c) RNA primers are needed to start new strands.

Synthesis of Eukaryotic DNA

The synthesis of DNA in eukaryotes is less well investigated than in bacteria. Nonetheless, the same general principles apply, although there are differences in detail from the bacterial scheme. In eukaryotes, semi-conservative replication occurs. One new strand is made continuously and the other in fragments. Both strands are made simultaneously by a replisome consisting of a helicase plus two DNA polymerase assemblies. A sliding clamp holds the polymerase on the DNA. An RNA primer is required.

In animal cells, two DNA polymerases (a and S) are involved in chromosome replication (Fig. 5.31). DNA polymerase a is responsible for initiation of new strands. It is accompanied by two smaller proteins that make the RNA primer. After the RNA primer has been made, polymerase a elongates it by a short piece of DNA only three or four bases long (the initiator DNA, or "iDNA").

Another protein, Replication factor C (RFC), then binds to the iDNA and loads DNA polymerase 8 plus its sliding clamp (PCNA protein) onto the DNA. Two assemblies of DNA polymerase S elongate the two new strands. The sliding clamp of animal cells is a trimer (not a dimer as in bacteria) that forms a ring surrounding the DNA. It was named PCNA, for proliferating cell nuclear antigen, before its role was fully known.

Linking of the Okazaki fragments differs significantly between animal and bacterial cells. In animals, there is no equivalent of the dual function polymerase I of bacteria. The RNA primers are removed by an exonuclease (MF1) and the gaps are filled by the DNA polymerase S that is working on the lagging strand. As in bacteria, the nicks are sealed by DNA ligase.

The presence of a nucleus complicates cell division in eukaryotes. The result is a complex cell cycle that includes dissolution and reassembly of the nucleus as well as duplication of the chromosomes.

Cell Division in Higher Organisms

The eukaryotic cells of higher organisms face further problems during cell division. Not only do they have multiple chromosomes, but these are inside the nucleus, separated from the rest of the cell by the nuclear membrane. Consequently, an elaborate process is needed to disassemble the nucleus, replicate the chromosomes and partition them among the daughter cells. This process is mitosis and involves several operations:

1. Disassembly of the nuclear membrane of the mother cell

2. Division of the chromosomes

3. Partition of the chromosomes

4. Reassembly of nuclear membranes around each of the two sets of chromosomes

5. Final division of the mother cell, or cytokinesis.

cytokinesis Cell division

DNA polymerase a Enzyme that makes short segment of initiator DNA during replication of animal chromosomes DNA polymerase 8 Enzyme that makes most of the DNA when animal chromosomes are replicated initiator DNA (iDNA) Short segment of DNA made just after the RNA primer during replication of animal chromosomes PCNA protein The sliding clamp for the DNA polymerase of eukaryotic cells (PCNA = proliferating cell nuclear antigen) replication factor C (RFC) Eukaryotic protein that binds to initiator DNA and loads DNA polymerase 8 plus its sliding clamp onto the DNA semi-conservative replication Mode of DNA replication in which each daughter molecule gets one of the two original strands and one new complementary strand

Cell Division in Higher Organisms 131

FIGURE 5.32 The Eukaryotic Cell Cycle

DNA replication occurs during the S phase of the cell cycle but the chromosomes are actually separated later, during mitosis or M phase. The S and M phases are separated by G1 and G2.

Mitosis itself is only one of several phases of the eukaryotic cell cycle (Fig. 5.32). The process of DNA replication described above takes place in the synthetic, or S-phase, of the cell cycle. The S-phase is separated from the actual physical process of cell division (mitosis) by two gap phases, or G-phases, in which nothing much appears to happen (except the normal processes of cellular activity and metabolism). Together, G1, S and G2 constitute interphase.

cell cycle Series of stages that a cell goes through from one cell division to the next

G1 phase Stage of the eukaryotic cell cycle following cell division;cell growth occurs here

G2 phase Stage of the eukaryotic cell cycle between DNA synthesis and mitosis: preparation for division interphase Part of the eukaryotic cell cycle between two cell divisions and consisting of G1-, S- and G2- phases mitosis Division of eukaryotic cell into two daughter cells with identical sets of chromosomes

S-phase Stage in the eukaryotic cell cycle in which chromosomes are duplicated

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