Prokaryotic replication Flashcards
Explain the mode of replication in E.coli
E.coli chromosome replicates by the bidirectional theta mode from a single replication origin.
What is the replisome
The E-coli replisome contains two Pol III holoenzyme complexes joined by a dimer of t(tau) subunits that bridges the alpha subunits. Both lagging and leading strand synthesis occurs at the replisome. Hence, the lagging strand must loop around to allow the extension of primosome-primed lagging strand.
What is the primosome
E.coli replication requires the participation of a complex protein assembly known as the primosome. Its formation is trigerred by the binding of PriA, followed by PriB and PriC. DnaB and DnaC proteins in the form of DnaB6.DnaC6 complex add to the DNA with the help of the DnaT protein in an ATP-requiring process. DnaC protein is then released and this yields the preprimosome. Then the preprimosome binds primase and this yields the primosome.
Explain DNA replication in E.coli
Duplex DNA is unwound by DnaB helicase on the lagging strand template, where it is joined by the primosome. The separated single strands are immediately coated with SSBs. Leading strand synthesis is catalyzed by Pol III holoenzyme, as is that of the lagging strand after priming by primosome-associated primase. Lagging and leading strand synthesis occurs on the replisome. After completing the synthesis of an Okazaki fragment, the lagging strand holoenzyme relocates to a new primer near the replication fork, the primer of the previously synthesized Okazaki fragment is excised by Pol I catalyzed nick translation and the nick is sealed by DNA ligase. Since lagging strand synthesis is more complex it takes more time than leading strand synthesis- the replisome coordinates this.
Explain the structure of oriC
The replication origin of the E-coli chromosome consists of a unique 250 bp segment called the oriC locus. It is highly conserved and supports bidirectional replication. The oriC locus contains 5 highly conserved 9 bp segments with consensus sequences known as DnaA boxes because they are bound by DnaA protein. The left boundary of oriC contains 3 repeated 13bp AT-rich segments known as DNA unwinding elements (DUEs).
Explain initiation of replication in E.coli
DnaA consists of 4 domains- a helicase interaction domain, a linker, an ATPase domain that is a member of the AAA+ family and a DNA binding domain. In the presence of ATP DnaA recruits additional DnaA subunits to form a right handed helix of DnaA subunits that is bound to the DNA. This generates local positive supercoils. The superhelical strain resulting from the compensating negative supercoils melts the DUE-containing segment. Alternatively or in addition the DnaAs ATPase domains may unwind the DNA. This process is facilitated by 2 DNA binding proteins- HU and IHF (Integration host factor). The oriC-DnaA complex recruits two DnaB6.DnaC6 complexes to the opposite ends of the melted region to form the prepriming complex. DnaC loads the the DnaB hexamers onto the DNA. In the presence of SSB and DNA gyrase, DnaB further unwinds the DNA. This allows the entry of primase and DNA polymerase.
Explain the regulation of the initiation of replication in E.coli
Chromosome replication in E-coli occurs only once per cell division. The doubling time of E.coli (cells) is from 20mins to 10 hours. E.colis chromosome replication time is about 40 mins (C). The segregation of cellular components is about 20 mins(D). If C+D is less than 60 mins then chromosome replication is initiated before the end of the preceding cell division cycle. This results in the formation of multiforked chromosomes. Even if there are multiple oriC sites present it can only be used once per cell generation. After initiation chain elongation proceeds at a uniform and uncontrolled rate. This suggests that post-initiation oriC site is somehow prevented from interacting with the replication machinery- a process called sequestration.
What does the fact that the E.coli chromosome is associated with the cell membrane explain
It helps to explain how replicated chromosomes are segregated into different cells during cell division
What is the mechanism of sequestration
The sequence most commonly methylated in E.coli is the palindrome GATC, which is methylated at both its A bases by Dam methyltransferase. GATC occurs 11 times in oriC, including at the beginning of all 3 of its 13 bp DUEs. Newly replicated GATC segments are hemimethylated- the GATC sequences on the newly synthesized strand are unmethylated. Although Dam methyltransferase begins methylating most hemimethylated GATC segments immediately after their synthesis, those on oriC remain hemimathylated for around a third of a cell generation. Hemimethylated GATCs on the oriC are bound to the membrane in a way that makes them inaccessible to both the initiation machinery and Dam methyltransferase.This association requires SeqA protein- it mediates sequestration by binding of hemimethylated oriC to membrane
Explain the structure of the E.coli replisome
The sliding clamp which is a ring shaped dimer through which the DNA that is replicated moves. Each replisome synthesizes around one Okazaki fragment per second- the sliding clamp must be loaded onto the lagging strand template at this rate. This process is ATP-dependent. The clamp loader must tightly bind the sliding clamp before its loading to the template DNA. The structures of the clamp loader and the beta-sigma complex explains how this occurs. The binding of ATP to gamma1 results in a conformational change that exposes the ATP-binding site of gamma2, the binding of ATP to gamma2 exposes gamma3 and ATP binding to gamma3 exposes the sigma subunits beta interaction element, thereby permitting it to bind to a beta subunit which opens the sliding clamp. The primer-template DNA then inserts itself through the gap in the sliding clamp. Eventually, the beta subunit and DNA stimulated hydrolysis of ATPs releases the beta subunit from the clamp loader which allows the sliding clamp to close around the DNA. The departure of the clamp loader permits Pol lII core to bind to the sliding clamp. When the synthesis of an Okazaki fragment is finished the Pol III core must dissociate- it releases the DNA and the sliding clamp. It then binds to a new primed template and clamp. The clamp that remains around the completed Okazaki fragment recruits Pol I and DNA ligase.
Explain the termination of replication in E.coli
The E.coli replication terminus is a large (350 kb) region that is flanked by 10 nearly identical nonpalindromic sites. The oriC is directly opposite the terminus region. A replication fork travelling counterclockwise stops on encountering TerA and a replication fork travelling clockwise stops as it encounters TerC. This allows the replication forks to enter the termination regions but not to leave and it guarentees that the two replication forks generated by bidirectional replication will meet in the replication terminus. The arrest of the replication fork requires a Tus protein- it binds to a Ter site where it prevents the displacement of the strands by DnaB. The Tus protein inhibits the helicase action of DnaB. Tus prevents the progress of DnaB in unwinding DNA from one side of Tus but not the other. The encounter of DnaB with a Tus-Ter complex in the permissive direction causes Tus to rapidly dissociate from the DNA, whereas the encounter of DnaB in the nonpermissive direction generates a locked Tus-Ter complex. This termination system is not essential for termination.
When the replication terminus is deleted, replication stops through the collision of opposing replication forks. As two oppositely moving replication forks collide at the termination site, the newly synthesized strands become covalently linked to yield two covalently closed double stranded chromosomes. However, since the parental DNA strands remain wound about each other about each other by several turns, the product dsDNA strands must be wound about each other by the same number of turns. The resulting catenated circular dsDNAs must be separated so that each can be passed to a different daughter cell- this is done by the type II topoisomerase names topoisomerase IV.
