DNA replication in E. coli

Leading and Lagging Strands Prepare a table that lists the names and compares the functions of the precursors, enzymes, and other proteins needed to make the leading versus lagging strands during DNA replication in E. coli. 

We look at E. coli DNA polymerase I as an example of how biochemical and genetic studies are used to characterize a DNA replication enzyme. Then we consider the other major proteins involved in DNA replication in E. coli before examining the proteins that participate in the synthesis of DNA in different types of organisms. 

DNA replication in E. coli starts at a unique origin (oriC) and proceeds sequentially in opposite directions. More than 20 proteins are required for replication. An ATP-driven helicase unwinds the oriC region to create a replication fork. At this fork, both strands of parental DNA serve as templates for the synthesis of new DNA. A short stretch of RNA formed by primase, an RNA polymerase, primes DNA synthesis. One strand of DNA (the leading strand) is synthesized continuously, whereas the other strand (the lagging strand) is synthesized discontinuously, in the form of 1-kb fragments (Okazaki fragments). Both new strands are formed simultaneously by the concerted actions of the highly processive... 

Comparison of Parameters of DNA Replication in E. coli and Human Cells... 

DNA SYNTHESIS IN PROKARYOTES DNA replication in E. coli has proven to be a complex process that consists of several basic steps. Each step requires certain enzyme activities. 

Despite the complexity of DNA replication in E. coli, as well as its rate (as high as 1000 base pairs per second per replication fork), this process is amazingly accurate (approximately one error per 109 to 1010 base pairs per generation). This low error rate is largely a consequence of the precise nature of the copying process itself (i.e., complementary base pairing). However, both pol III and pol I also proofread newly synthesized DNA. Most mispaired nucleotides are removed (by the 3 — 5 exonuclease activities of pol III and pol I) and then replaced. Several postreplication repair mechanisms also contribute to the low error rate in DNA replication. 

All the genetic information of bacteria such as E. coli is contained on a single circular piece of DNA made up of about three million nucleotide pairs and called the chromosome. DNA replication in E. coli begins at a unique sequence on the circular chromosome known as the replication origin. Replication occurs bidirectionally at the rate of about five hundred new nucleotides every second Because DNA synthesis occurs bidirectionally, there are two replication forks moving in opposite directions. Replication is complete when the two replication forks meet halfway around the circular chromosome. 

Nalidixic acid is an inhibitor of DNA replication in E. coli. It acts by binding to and inhibiting DNA gyrase. 

Match the functions or features related to DNA replication in E. coli listed in the right column with the molecules or structures in the left column. 

Mode-of-Action - Overlooked in the I96T review was a paper by Boyle, Goss and Cook on nalidixic acid I an alteration of normal DNA replication (in E. coli) occurs under its influence. In the new pattern, DNA synthesis occurs at new sites on the chromosomes, and requires concomitant protein and/or RNA synthesis. Pianotti, Mohan and Schwartz implicated DNA metabolism in the mechanism of action of the related oxollnic acid II. 

DNA replication in E. coli (Figure 20.6) has the following properties, (a) Replication starts at a unique site on the circular bacterial genome, called the origin of replication, (b) Replication proceeds simultaneously in both directions around the genome, that is, it is bidirectional. In other words, there are two replication forks, one travelling clockwise and one anticlockwise, at about the same rate, (c) Replication is terminated at a point diametrically opposite the origin of replication. 

Mammalian plasmid-based vectors, in addition to a prokaryotic replicon and a selection marker to permit DNA replication in E. coli, commonly have a eukaryotic replicon and a eukaryotic selection marker. The replicon usually comes from viruses such as Simian virus 40 (SV40), bovine papilloma virus (BPV) or Epstein Barr virus (EPV). The commonly used promoters are the SV40 early promoter (including its upstream enhancer... 

Dam mutants generally show a pleiotropic phenotype (39) they are hypermuta-ble, as are the strains that overproduce the MTase more than 10-fold (3,40) they are defective in their ability to restrict A DNA (41) and grow poorly in the presence of certain base analogs like 2-aminopurine and 5-Br-U (42). However, the combination of dam-3 with polA, recA, recB, or recC is lethal (38). Available evidence suggests that Dam plays a role in mismatch repair and in the control of DNA replication in E. coli (43-45). 

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