Lagging-strand synthesis

Elongation The elongation phase of replication includes two distinct but related operations leading strand synthesis and lagging strand synthesis. Several enzymes at the replication fork are important to the synthesis of both strands. Parent DNA is first unwound by DNA helicases, and the resulting topological stress is relieved by topo-isomerases. Each separated strand is then stabilized by... 

Lagging strand synthesis, as we have noted, is accomplished in short Okazaki fragments. First, an RNA... 

Many proteins are required for DNA synthesis and chromosomal replication. These include polymerases helicases, which unwind the parental duplex enzymes that fill in the gaps and join the ends in the case of lagging-strand synthesis enzymes that synthesize RNA primers at various points along the DNA tem-... 

In mammalian cells, at least eight DNA polymerases are present. DNA polymerase a is involved in the initiation of DNA synthesis at DNA replication origins and lagging strand synthesis (Wang, 1991). DNA polymerase 7is a mitochondrial DNA polymerase (Wang, 1991). Recently, bypass polymerases, such as DNA polymerase V, t. and Chave also been identified (Lindahl and Wood, 1999). These DNA polymerases are capable of continuing DNA synthesis even through bulky DNA lesions—such as UV-induced pyrimidine-dimers in the template strand (Lindahl and Wood, 1999). 

Telomerase is a ribonucleoprotein complex that exists in eukaryotic cells for the apparently sole purpose of synthesizing telomeric DNA, which consists of tandemly repeated sequences that contain clusters of G-residues and forms the ends of chromosomes. Telomerase comprises two essential core components, a protein subunit that has reverse transcriptase (RT) activity and an RNA sequence (hTR) that contains clusters of C-residues and serves as the template substrate for the RT (6). The G-rich DNA and C-rich RNA anneal to form a partial duplex with DNA as the primer. RT-mediated polymerization of dGTP and other complementary triphosphate substrates produces a DNA terminus that has been extended by around six nucleotides. The new end can become a substrate for either another round of telomerase-mediated elongation or primase/polymerase-mediated lagging-strand synthesis. 

Fig 11.16 SSBPs stabilise ssDNA and prevent it from re-annealing inappropriately. Primase synthesize the RNA primers required for initiating leading and lagging strand synthesis. 

Lagging strand synthesis is much more complex and involves five steps ... 

DNA polymerase then incorporates a dNMP onto the 3" end of the primer and initiates lagging strand synthesis. The polymerase extends the primer for about 1,000 nucleotides until it comes in contact with the 5 end of the preceding primer. These short segments of RNA/DNA are known as Okazaki fragments (Fig 11.24). 

Leading strand syntlresis can proceed all the way to the end of a chromosome however lagging strand synthesis can not. Consequently the 3 tips of each daughter chromosome would not be replicated. 

Figure 3-17. Mechanism of DNA synthesis at the replication fork. Two rounds of polymerase action are shown. The number of nucleotides added in each round is much larger than shown in eukaryotes, about 10 ribonucleotides and 200 deoxyribonucleotides are polymerized on the lagging strand. Synthesis on the leading strand is continuous.

Eukaryotic DNA polymerases have also been isolated and characterized as listed in Table 22.1. Based on studies of SV40 DNA replication in vitro, it has been found that DNA polymerase d has high processivity and is required for leading-strand synthesis, making it analogous to E. coli DNA pol III. DNA polymerase d requires ATP and is stimulated by two additional DNA replication proteins, RF-C and PCNA. DNA polymerase a serves the same role as E. coli DNA pol I in that DNA polymerase a is necessary for lagging-strand synthesis. In addition to DNA polymerase a and d, three other DNA polymerizing activities have been identified. DNA polymerase I is involved in DNA repair and is most similar to E. coli DNA pol II. DNA polymerase b is also a repair enzyme, and DNA polymerase g is required for mitochondrial DNA synthesis. 

Biochemical analysis of the large multiprotein complex used in T4 DNA replication revealed that it had a molecular weight in excess of 106 Daltons. In addition, it was determined that many of the proteins required for fork movement (Fig. 22.7), are actually tightly associated with each other in what has been called a replisome. One way to explain how the replisome can perform multitask functions such as lagging-strand synthesis, and still attain... 

Two key elements in this version of replisome-driven fork movement are that the DNA primase and helicase are tightly associated with each other, and that two molecules of the DNA pol III holoenzyme coordinately synthesize the new strands. The lagging-strand synthesis would still be discontinuous because the DNA pol III molecule on that strand would have to intermittently release and reattach as one Okazaki fragment was completed and before a second one was started. The DNA... 

Notable members comprising the Family B polymerases include the prototypical E. coli Pol II, as well as eukaryotic polymerases, Pol a, Pol S, Pol e, and Pol (. In humans, the coordinated efforts of Pol a, Pol 8, and Pol e are responsible for leading and lagging strand synthesis during nuclear chromosomal replication prior to... 

Distinctive for containing a primase activity, it is also highly sensitive to an inhibitor called aphidicolin. Functions in lagging strand synthesis. 

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