Chapter 15 DNA Biosynthesis and DNA Damage Repair,Section One The General Features of Replication of Chromosomal DNA,DNA replicates semiconservatively. . Watson and Crick predicted that DNA might semiconservatively replicate.,the hypothesis _ p _ d _ p _ p _ d _ p,. In 1958 Meselson and Stahl demons- trated the semiconservative nature of DNA replication in E coli.,The experiment: CsCl equilibrium gradient density ultracentri- fugation of 15N labeled E coli DNA.,The density of DNA was increased by labeling it with 15N, a heavy isotope of nitrogen. This was done by growing E coli 15 ge- nerations in a medium that contained 15NH4Cl as its only nitrogen source.,15N DNA was extracted and subjected to CsCl equilibrium gradient density ultracen- trifugation. The DNA band position was recorded. There was one band of 15N DNA.,The bacteria were transferred to an 14NH4Cl medium and grown for one generation. The DNA density was determined again. The position of the DNA band was compared with that of the 15N DNA .,What was the result ?,There was one DNA band. It had a lower density than 15N DNA because its position was above on that of 15N DNA. It was 15N/14N hybrid DNA.,After another generation growing in the 14NH4Cl medium the bacterial DNA density was determined. There were two DNA bands.,One half of the DNA was 14N DNA, and another half was hybrid DNA. In succeeding generations the ratio of 14N DNA to hybrid DNA increased gradually. The hybrid DNA became less and less,summary DNA replicates in a semiconservative man- ner. When the two parental strands sepa- rate, each serves as the template for making a new , complementary strand.,2. The point at which separation of the strands and synthesis of new DNA takes place is known as the replication fork. The replication fork is Y-shaped. Two arms (V) are separated strands which act as the template and DNA synthesis is actively taking place. The body (I) is the parental DNA.,3.DNA replication is usually bidirectional. .Replicon: Any piece which replicates as a single unit is called a replicon. All bacterial chromosomes and many phage and virus DNA molecules are circular and comprise single replicons.,In contrast eukaryotic chromosomes consist of multiple replicons. . Origin: The initiation within a replicon always occurs at a fixed point known as the origin.,.Terminus: In a circular replicon there is a single termination site roughly 180 opposite the unique origin.,Summary In a circular replicon replication begins from the fixed origin and forms two repli-cation forks. The two replication forks proceed bidirec- tionally away from the origin and the strands are copied as they separate until the terminus is reached.,4. DNA replication is semidiscontinuous. .The mechanism of DNA replication allows only for synthesis in a 53 direction. . The two strands of DNA are antiparallel.,Question How is the parental strand that runs 53 past the replication fork copied ? The answer is semidiscontinuous replica- tion.,At each replication fork one strand ( the lead- ing strand ), whose template runs 35 past the replication fork, is synthesized as one con- tinuous piece, while the other strand ( the lag- ging strand ), whose template runs 53 past the replication fork is made discontinuously as short fragments in the reverse direction.,These short fragments are called Okazaki fragments. They are joined by DNA ligase and form the lagging strand.,5. Origins contain short AT-rich repeat se- quences. . Prokaryotic and eukaryotic origins have common features: a. They consist of multiple unique short repeat sequences. b. These sequences are recognition and binding sites of multi-subunit initiation factors.,c. These sequences are usually AT- rich. . E coli s origin is called oriC. It is 254bp long and contains three 13-bp direct repeats and four 9-bp inverted re- peats.,6. DNA replication needs priming. . DNA polymerases cannot initiate DNA replication by starting a new DNA chain. They can only add nucleotides to the 3 end of an existent piece of RNA or DNA under the direction of the template. The existent piece of RNA or DNA are called primer.,. The leading strand and all Okazaki frag- ments are primed by synthesis of a short piece of RNA ( an RNA primer ), which is then elongated with DNA by DNA poly- merase. . There are also DNA priming or nucleotide priming.,7.Multi-enzymes and proteins participate in DNA replication. . Topoisomerases regulate the type and level of supercoiling of dsDNA. . Helicases unwind the dsDNA. . SSBs bind and stabilize the single DNA strand. . Primase synthesizes the RNA primer.,. DNA polymerases elongate DNA chains. . DNA ligase joins Okazaki fragments.,8. DNA replication is of high fidelity. . There are two types of replication errors. a. base ( nucleotide ) substitution. b. nucleotide insertion or deletion. . There are two types of error controls a. presynthetic e