DNA Replication Help
The hydrogen bonds linking base pairs together are relatively weak bonds. During DNA replication, the two strands separate along this line of weakness in zipperlike fashion (Fig. 3-10).
Each strand of the DNA molecule can serve as a template against which a complementary strand can form (according to the rules of specific base pairing) enzymes known as DNA polymerases. This mode of replication, in which each replicated double helix contains one original (parental strand) and one newly synthesized daughter strand, is referred to as semiconservative replication.At least three forms of DNA polymerase have been identified in prokaryotes and at least four in eukaryotes. All DNA polymerase enzymes add free nucleotides only to the 3' ends of existing chains, so that the chains will grow from their 5' ends toward their 3' ends. All three kinds of DNA polymerases can also degrade DNA in the 3' to 5' direction. Enzymes that degrade nucleic acids are called nucleases. If the enzyme cleaves nucleotides from the end of the chain it is called an exonuclease; if it makes cuts in the interior of the molecule it is termed an endonuclease. As long as deoxyribonucleotide precursors are present in even moderate amounts, the synthetic activity of a DNA polymerase is greatly favored over its degradation activity. During replication, incorrectly paired bases have a high probability of being removed by the exonuclease activity of the DNA polymerases before the next nucleotide is added. This is part of the "proofreading system" that protects the DNA from errors (mutations).
All DNA polymerases can extend existing polynucleotide chains only from their 30 ends; they cannot initiate new chains from their 5' ends. A special kind of RNA polymerase, called primase, constructs a short (about 10 bp) segment of ribonu-cleotides (as an RNA primer) complementary to the DNA template at a specific DNA sequence called an origin of replication (ori) site. This RNA primer has a free 3' end to which additional deoxyribonucleotides can be added by DNA polymerases. Primers are later removed by aDNAnuclease enzyme. There may be one or many ori sites, depending on the type of DNA being replicated. For example, bacterial circular chromosomes have a single origin, while linear eukaryotic chromosomes have multiple origin sites. Each unit of replication is called a replicon. When multiple origin sites are present, they result in the formation of multiple replication bubbles during DNA replication (Figure 3-11).
Replication begins at the 30 end of a template (parental) strand (Fig. 3-12). A primer RNA is synthesized from 5' to 3' toward the replication fork, and the primer is extended by DNA polymerase III, forming the leading strand. The opposite template strand has a 5' end, so no complementary primer can be formed from 3' to 5'. Instead, a lagging strand is replicated (5' to 3') in short segments (a few hundred nucleotides each) in a direction opposite to the movement of the replication fork. These segments are called Okazaki fragments. A gap exists where one or more adjacent nucleotides are missing from one strand of a duplex DNA molecule. DNA polymerase I temporarily creates gaps by removing RNA primers, but quickly fills the gaps with replacement deoxyribonucleotides. Nicks between adjacent Okazaki fragments are rapidly joined by DNA ligase so that at any given time there is only a single incomplete fragment in the lagging strand. The discontinuous replication of the lagging strand results in its seemingly paradoxical overall growth from 3' to 5'.
At least two other classes of enzymes are also required for DNA synthesis. The helicases (unwinding proteins) proceed ahead of the DNA polymerases, opening the double helix and producing single-stranded templates for replication. These single-stranded regions are stabilized when complexed with single-stranded DNA binding proteins (SSB), forming a replication fork.
Two regions of eukaryotic chromosomes require special attention during replication: (1) the ends (called telomeres) and (2) the centromeres. The ends of eukaryotic chromosomes are maintained by special ribonucleoprotein enzymes (called telomerases) that add new terminal DNA sequences to replace those lost during each replication cycle.
EXAMPLE 3.2 The ends of the linear chromosomes in the macronucleus of the ciliated protozoan Tetrahymena have 30-70 tandemly repeated blocks of the sequence
A special enzyme adds 5'-TTGGGG-3' to the 30 end of any such sequence in single-stranded DNA. After the enzyme has extended the 3' end of the telomere, synthesis of the repeats in the complementary DNA strand could be primed by a primase. Similarly, all yeast chromosomes end with approximately 100 bp of the irregularly repeated sequence
The mechanism that determines the length of such telomeres is not well understood.
In the ciliate Euplotes crassus, the RNA component of the telomerase serves a template function for synthesis of telomeric T4G4 repeats. Thus, at least some portions of certain eukaryotic DNA molecules are known to be replicated from RNA templates. Enzymes that make DNA from RNA templates are called reverse tran-scriptases; this kind of telomerase represents a specialized kind of reverse tran-scriptase.
The centromeric regions of chromosomes play an important role in chromosome segregation. They are the site upon which spindle fibers attach to chromosomes to help them move. The fibers assemble on a proteinaceous structure called the kinetochore, which forms at the centromeres of each sister chromatid during late prophase. Centromeres are comprised of specific DNA sequences. For example, the centromeric sequences in yeast are about 120-130 bp long and contain two highly conserved sequences of 10-15 bp each that flank a 90 bp region that is very rich in A-T. The centromeres of animals and plants are larger and have more complex, highly repetitive nucleotide sequence structures. The chromatin material in the centromere region is not organized into nucleosomes but into highly condensed heterochromatin. This organization makes it difficult to determine the sequence. Also, the centromeres are the last sequences of DNA to be replicated during the cell cycle.
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