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Replication Fork | Science Primer

Elongation of the DNA chain on the lagging strand during DNA replication is discontinuous: Short segments of DNA, called Okazaki Fragments, are repeatedly synthesized in the reverse direction of movement of the replication fork (1). This occurs because the two chains of double-helical DNA are antiparallel, and DNA polymerase can extend a DNA chain only in thedirection. On the leading strand, which runsin the reverse direction to fork movement, the replicative enzyme carries out chain elongation continuously in a highly processive manner. On the other parent strand, the lagging strand, which runsin the direction of fork movement, DNA polymerase catalyzes chain elongation only in the reverse direction of fork movement. Thus, as the replication fork proceeds, the unreplicated segment is expanded on the lagging strand. When one act of chain elongation on the lagging strand is accomplished, the next round of chain elongation must be started from a newly expanded segment on the lagging strand. To achieve completion of DNA replication on an entire region of the lagging strand, numerous enzymes cooperate at the replication fork. They are participating in primer synthesis (the initiation of Okazaki fragments), chain elongation (the extension of Okazaki fragments), and a process that connects Okazaki fragments.

DNA polymerase III holoenzyme - Wikipedia

The chain elongation of Okazaki fragments in E. coli is catalyzed by DNA polymerase III holoenzyme (10). This enzyme possesses a capacity to synthesize DNA with a very high processivity, sufficient for completion of about 2 kb of Okazaki fragment. In addition, the Pol III holoenzyme dissociates from the nascent Okazaki fragment and restarts the next round of Okazaki fragment synthesis from an RNA primer newly settled near the replication fork (11). Enzymes to remove primer RNA and fill the gap, such as ribonuclease H and DNA polymerase I of E. coli, are essential for the sealing of Okazaki fragments by DNA ligase (12). Mutants defective in either DNA polymerase I or DNA ligase show a massive accumulation of short Okazaki fragments under restrictive conditions.

Why Is The Lagging Strand Synthesized In A ..

Discontinuous DNA Synthesis

In E. coli, there are two pathways by which the DnaB helicase is loaded onto the lagging-strand template DNA: One is primosome formation directed by the PriA protein, and the other is DnaA protein-directed DnaB loading (8, 9). The former process was discovered first in the replication of bacteriophage fX174 DNA and plasmid ColE1 DNA, and it later appeared to be involved in the resumption of chromosomal DNA replication after replication of the E. coli genome has been interrupted or halted. On the other hand, the latter process was found in oriC plasmid DNA replication in vitro and is thought to form a priming complex with DnaG primase at the replication fork in E. coli chromosomal DNA replication.

Although the basic biochemical processes that occur at eukaryotic and prokaryotic replication forks are similar, there are many differences in detail (13). For example, primer synthesis in eukaryotic cells is catalyzed by DNA polymerase a, which synthesizes 2 to 12 nucleotides of RNA (initiator RNA) and further adds about 20 nucleotides of DNA to the initiator RNA. The size of Okazaki fragments (40 to 300 nucleotides) in eukaryotes is significantly shorter than those observed in prokaryotes.

DNA Replication- Leading vs Lagging Strand - YouTube


ATM and ATR are both protein kinases that phosphorylate consensus SQ/TQ motifs in target proteins, including the histone H2AX. Phosphorylated H2AX (γH2AX) helps to recruit DNA repair proteins and promotes the activation of Chk1 and Chk2, leading to cell cycle arrest. Activation of ATM is triggered by DNA double-strand breaks (DSBs), oxidative damage, and alterations in chromatin structure, while RPA-bound single-stranded DNA activates ATR. Prior studies had shown that SV40 can replicate in the absence of functional ATM or ATR, but the result is a reduction in unit-length viral DNA. The Fanning lab’s goal was to understand how this occurs, and to determine the role of these two DDR proteins in SV40 replication. First, they infected monkey kidney BSC40 cells with SV40 virus and used immunofluorescence microscopy to observe the cellular localization of replication-related proteins (Figure 2). They found discrete regions in the nuclei of infected cells that labeled brightly with antibodies against viral Tag. These regions were also labeled by probes directed against ethynyl-2′-deoxyuridine (EdU), which had been incorporated into newly synthesized DNA. The host proteins Pol δ, RFC-1, and proliferating cell nuclear antigen (PCNA), all known to be involved in SV40 replication, co-localized with Tag and EdU. In contrast, Tag did not co-localize with Cdc45, which plays a role in host cell, but not viral DNA replication. These initial results indicated that labeling of Tag could be used to identify sites of SV40 DNA replication within the cells.

Due to the functional restriction of the not being able to synthesize the chain in 3’ to 5’ direction, on the lagging strand, the synthesis of the chain is discontinuous in the 5’ to 3’ direction. The discontinuous replication results in several short segments which are called .

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lagging strand replication during DNA synthesis

DNA polymerase cannot replicate duplex DNA without assistance. This enzyme requires single-stranded DNA as a template and an RNA or DNA primer annealed to the template. Two other enzymes enable polymerase to work on duplex DNA. One is a DNA helicase that opens up the duplex at the replication fork to provide a single-stranded template. The other is a primase that synthesizes a short RNA to prime DNA chain elongation. Several DNA helicases have been identified from E. coli and its phage, including DnaB, T7 gp4, and T4 gp41 (3-5). The biochemical characterization of these activities in in vitro DNA replication systems suggests that the primary replicative helicase binds to and moves on the lagging-strand template in thedirection, unwinding the DNA double helix as it goes. Another common property of the replicative helicases is an intimate association with a primase. The bacteriophage T7 gp4 has both primase and helicase activity within the same polypeptide chain (4). Bacteriophage T4 gp41 greatly enhances the primase activity of T4 gp61 (6). A similar functional interaction has been observed between DnaB protein and E. coli primase, DnaG protein (7). On most templates, DnaG exhibits a very feeble priming activity that can be greatly enhanced if DnaB first binds that DNA. This stimulation of primase activity is further increased when the DnaB helicase is activated to its processive form at the replication fork.

Discontinuous leading-strand synthesis: a stop–start …


To further understand the role of ATM in SV40 replication, the Fanning lab used two-dimensional agarose gel electrophoresis to examine the structure of replication intermediates formed in the presence and absence of Ku-55933. Theta replication produces structures known as Cairns intermediates (Figure 1B and 5). Cutting these intermediates using restriction enzymes can produce either a double Y structure or a bubble structure, depending on the location of the cut. Accordingly, BgII, which cleaves at the origin of replication, produced double Y structures from SV40 replication intermediates produced by infected cells in the absence of Ku-55933, while BamHI, which cleaves at the opposite side of the circle, produced bubble structures. However, when the same enzymes were used to cleave intermediates formed by infected cells in the presence of Ku-55933, a substantial quantity of simple Y structures and X structures were obtained in addition to more complex D-loop structures. These results suggested that, in the presence of Ku-55933, the SV40 genome was undergoing rolling circle replication, as opposed to its usual theta replication (Figure 5). Rolling circle replication proceeds around the circular viral template in one direction and can easily lead to the concatemeric structures produced in Ku-55933-treated SV40-infected cells.

Discontinuous lagging strand DNA synthesis at …

In Escherichia coli, more than 20 different proteins participate in DNA replication (2). These were identified by screening mutants defective in DNA replication and by purifying enzymes required for in vitro DNA synthesis (see dna genes). From their biochemical roles at different stages of chromosomal DNA replication, it appears that at least eight proteins are involved in the discontinuous replication in E. coli. Those are (1) primosome proteins, including DNA helicases and primase: PriA, PriB, PriC, DnaT, DnaB, DnaC, and DnaG (primase); (2) proteins required for chain elongation: DNA polymerase III (Pol III) holoenzyme and single-stranded DNA binding protein (SSB); and (3) proteins required for connecting Okazaki fragments: DNA polymerase I (Pol I), RNaseH, and DNA Ligase. Among these proteins, the DnaB helicase, primase (DnaG), and Pol III holoenzyme are the basic components acting on the discontinuous DNA synthesis at the replication fork, probably forming a multiprotein complex called a replisome.

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