The text under the title „DNA replication “written by Brooker analyses the process in which existing DNA strands are used to make new ones. In this text author explains how the structures of DNA are involved in this process by overviewing bacterial DNA replication and some interesting features of eukaryotic DNA replication.
First chapter presents the basic characteristics of DNA replication. Process begins when two complementary strands unwind and each parental strand is used as template to synthesize new daughter strands according to the AT/GC rule. Those strands are identical so they contain the same information. According to the author, some scientists in the late 1950s thought of three models of DNA replication: conservative when parental strands stay together, semiconservative when the new DNA strand consists of one newly made strand and one old strand and dispersive model when DNA contains segments of both parental and newly made strands. Meselson and Stahl proved that DNA replication is semiconservative with an experiment where they labeled DNA with heavy and light isotopes of nitrogen and used centrifugation.
Second chapter examines how DNA replication occurs within bacteria. Synthesis begins at a single origin of replication and continues bidirectionally until two replication forks meet each other. Distinction is made between different types of proteins that are involved in E. coli DNA replication. To initiate the replication, DnaA proteins bind to five DnaA box sequences at the origin and unwind the AT-rich region. Following that, two DNA helicases bind to single-stranded DNA and move in a 5’ to 3’ direction to maintain the replication fork moving. In front of DNA helicase travels DNA gyrase which alleviates positive supercoiling. The author points out that in order to keep the DNA strands separated replication requires single-strand binding proteins. At the next step of replication primase synthesizes RNA primers, which have the leading strand and the lagging strand, and DNA polymerase synthesizes complementary strands of DNA. As there are five types of DNA polymerase, the author notes that polymerase III is responsible for the most of the DNA replication. Several features are enumerated. Firstly, DNA polymerase needs an RNA primer to start the synthesis. Secondly, it can attach nucleotides only in the 5’ to 3’ direction. Because of these two features, there are some differences between the synthesis of leading strand and lagging strand. The leading strand is made continuously in the direction of the replication fork while the lagging strand consists of short Okazaki fragments and is made in the direction away from the fork. To continue the replication, the RNA primers have to be removed by DNA polymerase I, which later synthesizes DNA to fill the empty area. Then DNA ligase completes the replication by catalyzing a covalent bond between fragments. Later the text presents the main facts about enzymatic features of DNA polymerase. It is a processive enzyme that uses deoxynucleoside triphosphates to synthesize new DNA strands. When replication comes to an end it is terminated at the pair of termination sequences, called ter sequences, where oppositely moving forks meet. The author draws attention that often DNA replication results in two intertwined catenanes that need to be unlocked by topoisomerase II.
In the next paragraph it is explained that some enzymes bind to each other to form a complex. For example, the primosome is a complex between helicase and primase while the replisome is a complex between the primosome and dimeric DNA polymerase. Even though DNA replication proceeds very fast, the synthesis occurs with high fidelity. Firstly, it is result of stability of hydrogen bonding between the correct bases. Secondly, because of proofreading ability of DNA polymerase. To avoid mistakes in bacterial cell division DNA replication is controlled by the amount of DnaA protein and by methylation of GATC sites in origin. Further follows the example illustrating an experiment with Kornberg’s method to measure DNA replication in vitro. Another experiment which was useful for identifying proteins involved with DNA replication was the isolation and characterization of mutants.
Third chapter analyses eukaryotic DNA replication which is more complex than bacterial. For example, eukaryotic chromosomes contain not just one, but multiple origins of replication. Part of prereplication complex is formed from a group of six proteins called ORC. They initiate DNA replication. The binding of MCM helicase completes DNA replication licensing. The author points out that eukaryotes have different types of DNA polymerases with specific functions. They switch with each other during DNA replication. Another difference between bacterial and eukaryotic DNA replication is how RNA primers are removed. In eukaryotic replication enzyme called flap endonuclease is responsible for that. The ends of linear chromosomes contain telomeres and they are replicated by telomerase. It prevents chromosome shortening.
To sum up, in these three chapters the author introduced the basics of DNA replication. Later he pointed out the main differences between the bacterial DNA replication and eukaryotic DNA replication. Readers learnt that despite many similarities between them, eukaryotic DNA replication is more difficult and complex than bacterial.