coli culture was then shifted into a medium containing 14N and allowed to grow for one generation. coli for several generations in a medium containing a “heavy” isotope of nitrogen ( 15N) that was incorporated into nitrogenous bases and, eventually, into the DNA. Matthew Meselson (1930–) and Franklin Stahl (1929–) devised an experiment in 1958 to test which of these models correctly represents DNA replication ( Figure 11.5). In the dispersive model, all resulting DNA strands have regions of double-stranded parental DNA and regions of double-stranded daughter DNA. In the semiconservative model, parental strands separated and directed the synthesis of a daughter strand, with each resulting DNA molecule being a hybrid of a parental strand and a daughter strand. In the conservative model, parental DNA strands (blue) remained associated in one DNA molecule while new daughter strands (red) remained associated in newly formed DNA molecules. There were two competing models also suggested: conservative and dispersive, which are shown in Figure 11.4.įigure 11.4 There were three models suggested for DNA replication. In one model, semiconservative replication, the two strands of the double helix separate during DNA replication, and each strand serves as a template from which the new complementary strand is copied after replication, each double-stranded DNA includes one parental or “old” strand and one “new” strand. Separating the strands of the double helix would provide two templates for the synthesis of new complementary strands, but exactly how new DNA molecules were constructed was still unclear. The elucidation of the structure of the double helix by James Watson and Francis Crick in 1953 provided a hint as to how DNA is copied during the process of replication. Explain the process of rolling circle replication.Identify the differences between DNA replication in bacteria and eukaryotes.Describe the process of DNA replication and the functions of the enzymes involved.Explain why Okazaki fragments are formed.Explain why DNA replication is bidirectional and includes both a leading and lagging strand.Explain the meaning of semiconservative DNA replication.Sites of termination are, therefore, indicative of origin firing time – allowing us to faithfully reconstruct the temporal dynamics of the replication program using data from a single asynchronous culture.By the end of this section, you will be able to: Contrary to expectation, we find that centromeres and highly transcribed regions are not strong determinants of replication termination rather, termination generally occurs midway between two adjacent replication origins at positions dictated by the relative firing times of those origins. We provide evidence that S-phase follows a distinct temporal program dominated by replication origins firing with high probability within distinct time intervals. Our methodology also provides a genome-wide view of DNA replication: allowing the first detailed measurements of the efficiencies of all replication origins and regions of termination. Using deep sequencing, we have found that Okazaki fragment synthesis is strongly influenced by nucleosomes as well as certain other DNA bound factors. These Okazaki fragments are typically less than 500bp in length and are generally sized according to the nucleosome repeat. Recently, using transient DNA ligase I inactivation, we have been able to capture and analyze short DNA molecules produced on the lagging strand. Synthesis of the lagging strand occurs discontinuously and necessitates the repeated production of Okazaki fragments. DNA replication is inherently asymmetric.
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