Bacterial Dna Replication Proteins And Their Functions – Home » Student Resources » Online Chemistry Textbooks » CH450 and CH451: Biochemistry – Defining Life at the Molecular Level » Chapter 9: DNA Replication
The elucidation of the structure of the double helix by James Watson and Francis Crick in 1953 provided clues as to how DNA is copied during the process of DNA replication. The separation of the strands of the double helix provides two templates for the synthesis of new complementary strands, but it was still unclear how new DNA molecules were constructed. In one model, semiconservative replication separates from the two strands of the double helix during DNA replication, and each strand serves as a template from which a new complementary strand is copied. In this model, after replication, each double-stranded DNA consists of a parent or “old” strand and a daughter or “new” strand. There were also two competing models proposed: conservative and dispersive, shown in Figure 9.1.
Bacterial Dna Replication Proteins And Their Functions
Figure 9.1 Three models of DNA replication. In the conservative model, the parent DNA strands (blue) remained connected in one DNA molecule, while the new daughter strands (red) remained connected in the nascent DNA molecules. In the semiconservative model, the parent strands separated and directed the synthesis of the daughter strand, resulting in each DNA molecule being a hybrid of the parent strand and the daughter strand. All DNA strands formed in the dispersive model have double-stranded parent DNA regions and double-stranded daughter DNA regions.
Rna Holds The Reins In Bacteria: U M Researchers Observe Rna Controlling Protein Synthesis
Matthew Meselson and Franklin Stahl devised an experiment in 1958 to test which of these models correctly represented DNA replication (Figure 9.2). They grew several generations of Escherichia coli bacteria in a medium containing a “heavy” isotope of nitrogen (15N) incorporated into nitrogenous bases and eventually DNA. This labeled the parental DNA. The E. coli culture was then transferred to medium containing 14N and allowed to grow for one generation. Cells were harvested and DNA was isolated. DNA was separated by ultracentrifugation, during which bands formed according to DNA density. DNA grown at 15N is expected to form a band at a higher density position than that grown at 14N. Meselson and Stahl noted that the single band observed after one generation of growth on 14N was at an intermediate position between the DNA of cells grown only on 15N or 14N. This suggested either a semiconservative or a dispersive mode of replication. Some cells were allowed to grow for one more generation in 14N and spun again. DNA collected from cells grown for two generations in 14N produced two bands: one DNA band was intermediate between 15N and 14N, and the other corresponded to the 14N DNA band. These results can only be explained by semiconservative DNA replication. Therefore, the other two models were excluded. As a result of this experiment, we already know that during DNA replication, each of the two strands forming a double helix serves as a template from which new strands are copied. The new thread will complement the parent or “old” thread. The resulting DNA molecules have the same sequence and divide equally into two daughter cells.
Figure 9.2 Meselson and Stahl experimented with E. coli grown first in heavy nitrogen (15N) and then in 14N. DNA grown in 15N (blue lane) was heavier than DNA grown in 14N (red lane) and precipitated to a lower level in ultracentrifugation. After one round of replication, the DNA collapsed halfway between the 15N and 14N levels (purple band), ruling out a conservative replication model. After the second round of replication, a dispersive model of replication was excluded. These data supported a semiconservative replication model.
DNA replication has been well studied in bacteria, primarily due to the small size of the genome and the mutants available. E. coli has 4.6 million base pairs (Mbp) on a single circular chromosome, all of which replicate in about 42 minutes, starting from a single origin of replication and proceeding bidirectionally (ie, in both directions) around the circle (Figure 9.3). ). This means that about 1000 nucleotides are added per second. The process is quite fast and occurs with few errors. One chromosome of E. coli has a single origin of replication called oriC. The origin of replication is approximately 245 base pairs long and rich in adenine-thymine (AT) sequences.
Figure 9.3 Prokaryotic DNA Replication. In prokaryotes, DNA replication begins at a single origin of replication, shown in the figure on the left, and proceeds bidirectionally around a circular chromosome until replication is complete. The bidirectional nature of replication creates two replication forks that actively mediate the replication process. The figure on the right shows the dynamic model of this process. Red and blue dots represent the incorporation of daughter strand nucleotides during the replication process.
Lecture 24: Dna Replication
Open regions of DNA that are actively being replicated are called replication forks. All the proteins involved in DNA replication assemble at replication forks to form a replication complex called a replisome (Table 9.1 and Figure 9.4). DNA replication in the model organism E. coli has been extensively studied, providing a basis for understanding the various mechanisms of genome duplication used by all organisms. DNA replication in E. coli begins at orC (Figure 9.3). oriC “melts” under the action of the DnaA initiator protein to expose two template ssDNA strands that act as a platform for loading the replicative DnaB helicase. One complete DnaB hexamer is loaded onto each ssDNA strand with the help of the helicase loader DnaC. Additional exposed ssDNA is quickly coated with ssDNA-binding protein (SSB), which protects the DNA and blocks additional DnaB helicase loading. Each DnaB hexamer recruits primase (DnaG), which synthesizes the RNA primers used to initiate DNA synthesis, along with the subunits that make up the replicative DNA polymerase III holoenzyme (PolIII HE). These proteins form the core replisomes that copy the E. coli genome. After assembly, replisomes bidirectionally replicate from orC until they encounter regions bound by Tus proteins, ideally forming “replication fork traps,” where they undergo programmed disassembly at the end region. After DNA replication is complete, the newly synthesized genomes are separated and segregated into daughter cells.
Figure 9.4 Overview of the DNA Replication Fork. At the start of replication, topoisomerase II relaxes the hypercoiled chromosome. Two replication forks are formed by the unwinding of double-stranded DNA at the origin, and helicase separates the DNA strands, which are coated with single-stranded binding proteins to keep the strands apart. DNA replication occurs in both directions. An RNA primer complementary to the parental strand is synthesized by RNA primase and elongated by DNA polymerase III by adding nucleotides to the 3′-OH terminus. In the leading strand, DNA is synthesized continuously, while in the lagging strand, DNA is synthesized in short pieces called Okazaki fragments. RNA primers within the lagging strand are removed by the exonuclease activity of DNA polymerase I, and Okazaki fragments are joined by DNA ligase.
As mentioned above, bacterial chromosome replication is initiated at oriC, where the initiator protein, DnaA, binds to initiate assembly of the enzymatic replisome machinery. The initial steps in this process involve the assembly of the primosome, which functions to unwind the two strands of DNA at the replication fork and add RNA primers to the DNA templates to be used by DNA Polymerase enzymes to initiate replication. After DnaA-induced replication origin remodeling, bacterial loader-dependent primosome assembly occurs in discrete steps and involves the coordinated action of at least four different proteins (initiation protein, helicase, helicase loading protein, and primase). and consistently (Table 9.1).
The oriC region of prokaryotes contains highly conserved sequence motifs, including an AT-rich box domain that serves as a recognition sequence for binding of the DnaA initiator protein. The initial binding of DnaA to oriC promotes the unwinding of the DNA double helix and the assembly of multiple DnaA subunits forming a helical oligomer along the newly unwound single-stranded DNA (ssDNA) (Figure 9.5). The DnaA protein has four main domains. Domains III and IV are integral to binding ssDNA, while domain I is involved in protein-protein interactions. Domain II forms a flexible linker between the protein interaction domain and the DNA binding domains.
Solved Draw A Diagram Of The Dna Replication In Bacteria
Figure 9.5 Primosome assembly. Melting of DNA at oC and loading of the DnaB6-(DnaC)6 helicase-loader complex into a DNA vesicle. Bottom schematic: ATP-bound DnaA (initiation protein) binds to DnaA boxes via Domain IV, thereby promoting dsDNA wrapping around the DnaA filament and causing torsional stress in the dsDNA. At the same time, Domain III of DnaA binds to one of the two ssDNA strands of the DNA unwinding element and elongates the strand. These interactions cause melting of the AT-rich DNA unwinding element and bubble formation. At the same time, binding of DnaC (the helicase loader) keeps DnaB (the helicase) in an open lock washer conformation to allow loading onto ssDNA. DnaC interacts with DnaA at the end of the filament