Chapter Summary

Chapter Summary

14.1 Historical Basis of Modern Understanding

DNA was first isolated from white blood cells by Friedrich Miescher, who called it nuclein because it was isolated from nuclei. Frederick Griffith's experiments with strains of Streptococcus pneumoniae provided the first hint that DNA may be the transforming principle. Avery, MacLeod, and McCarty proved that DNA is required for the transformation of bacteria. Later experiments by Hershey and Chase using bacteriophage T2 proved that DNA is the genetic material. Chargaff found that the ratio of A = T and C = G, and that the percentage content of A, T, G, and C is different for different species.

14.2 DNA Structure and Sequencing

The currently accepted model of the double-helix structure of DNA was proposed by Watson and Crick. Some of the salient features are that the two strands that make up the double helix are complementary and anti-parallel in nature. Deoxyribose sugars and phosphates form the backbone of the structure, and the nitrogenous bases are stacked inside. The diameter of the double helix, 2 nm, is uniform throughout. A purine always pairs with a pyrimidine; A pairs with T, and G pairs with C. One turn of the helix has 10 base pairs. During cell division, each daughter cell receives a copy of the DNA by a process known as DNA replication. Prokaryotes are much simpler than eukaryotes in many of their features. Most prokaryotes contain a single, circular chromosome. In general, eukaryotic chromosomes contain a linear DNA molecule packaged into nucleosomes, and have two distinct regions that can be distinguished by staining, reflecting different states of packaging and compaction.

14.3 Basics of DNA Replication

The model for DNA replication suggests that the two strands of the double helix separate during replication, and each strand serves as a template from which the new complementary strand is copied. In conservative replication, the parental DNA is conserved, and the daughter DNA is newly synthesized. The semi-conservative method suggests that each of the two parental DNA strands acts as template for new DNA to be synthesized; after replication, each double-stranded DNA includes one parental or old strand and one new strand. The dispersive mode suggested that the two copies of the DNA would have segments of parental DNA and newly synthesized DNA.

14.4 DNA Replication in Prokaryotes

Replication in prokaryotes starts from a sequence found on the chromosome called the origin of replication—the point at which the DNA opens up. Helicase opens up the DNA double helix, resulting in the formation of the replication fork. Single-stranded binding proteins bind to the single-stranded DNA near the replication fork to keep the fork open. Primase synthesizes an RNA primer to initiate synthesis by DNA polymerase, which can add nucleotides only in the 5' to 3' direction. One strand is synthesized continuously in the direction of the replication fork; this is called the leading strand. The other strand is synthesized in a direction away from the replication fork, in short stretches of DNA known as Okazaki fragments. This strand is known as the lagging strand. Once replication is completed, the RNA primers are replaced by DNA nucleotides and the DNA is sealed with DNA ligase, which creates phosphodiester bonds between the 3'-OH of one end and the 5' phosphate of the other strand.

14.5 DNA Replication in Eukaryotes

Replication in eukaryotes starts at multiple origins of replication. The mechanism is quite similar to prokaryotes. A primer is required to initiate synthesis, which is then extended by DNA polymerase as it adds nucleotides one by one to the growing chain. The leading strand is synthesized continuously, whereas the lagging strand is synthesized in short stretches called Okazaki fragments. The RNA primers are replaced with DNA nucleotides; the DNA remains one continuous strand by linking the DNA fragments with DNA ligase. The ends of the chromosomes pose a problem as polymerase is unable to extend them without a primer. Telomerase, an enzyme with an inbuilt RNA template, extends the ends by copying the RNA template and extending one end of the chromosome. DNA polymerase can then extend the DNA using the primer. In this way, the ends of the chromosomes are protected.

14.6 DNA Repair

DNA polymerase can make mistakes while adding nucleotides. It edits the DNA by proofreading every newly added base. Incorrect bases are removed and replaced by the correct base, and then a new base is added. Most mistakes are corrected during replication, although when this does not happen, the mismatch repair mechanism is employed. Mismatch repair enzymes recognize the wrongly incorporated base and excise it from the DNA, replacing it with the correct base. In yet another type of repair, nucleotide excision repair, the incorrect base is removed along with a few bases on the 5' and 3' end, and these are replaced by copying the template with the help of DNA polymerase. The ends of the newly synthesized fragment are attached to the rest of the DNA using DNA ligase, which creates a phosphodiester bond.

Most mistakes are corrected, and if they are not, they may result in a mutation defined as a permanent change in the DNA sequence. Mutations can be of many types, such as substitution, deletion, insertion, and translocation. Mutations can be induced or may occur spontaneously.