Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies
to form a transient but extended RNA–DNA hybrid (called R-loop) (Figure 2.9). This would displace a single strand of DNA that provides a prime target for loading of the helicase/primase complex, with subsequent loading and activation of both leading- and lagging-strand DNA polymerases (Figure 2.9). This would constitute a complete replisome like the ones discussed above, traveling away from the origin (in the rightward direction in Figure 2.9). Note that the lagging-strand DNA polymerase travels with the rightward replisome complex, as depicted by the dotted arrow in Figure 2.9 (and would actually be associated with that complex as discussed above). This initiation process has been recreated in vitro and requires the four T7 replication proteins along with the T7 RNA polymerase.
With the folding of the lagging strand around into a loop structure, one might expect that the replication-origin region would contain special features that create this loop. However, this is not the case. Very simple DNA substrates such as the one diagrammed in Figure 2.9 are converted into properly folded lagging-strand loops by the simple T7 replication machinery discussed above. Thus, the replisome complex discussed above has the intrinsic ability to create properly folded lagging-strand loops.
DNA replication from the T7 origin occurs in a “bidirectional” fashion, with one replisome generated in each of the two directions from the origin. Thus, after the first replisome is assembled and travels away from the origin as described above (the rightward direction in Figure 2.9), a second replisome complex is assembled for the leftward direction. In this case, the 3′ end of the first Okazaki fragment from the rightward direction is used as the primer for the new leading strand in the leftward direction (Figure 2.9). The precise pathway of protein loading for leftward direction is not yet clear, but the combination of a branch with a free 3′ end (from the first rightward Okazaki fragment) appears to be sufficient to trigger loading.
2.10Summary of key points
•The bacteriophage T7 replisome consists of only four proteins: the helicase/primase, ssDNA-binding protein, and DNA polymerase with its host thioredoxin partner.
•T7 DNA polymerase has separate active sites for polymerization and proofreading exonuclease activities.
•Proofreading exonuclease greatly reduces the mutation rate by removing a large majority of misinserted nucleotide residues.
•Processivity of T7 DNA polymerase is increased by the thiore-doxin protein partner, interaction with the T7 helicase/primase, and rapid rebinding of polymerase when it dissociates from the 3′ end.
•T7 helicase/primase unwinds DNA by translocating along a single-strand in a “hand-over-hand” manner, whereby each subunit of the hexamer shifts sequentially from the back to the front of the lock-washer-like structure.
•T7 DNA polymerase stimulates the unwinding activity of the helicase/primase complex, and thus the two proteins are mutually reinforcing.
•The helicase/primase complex travels along the lagging-strand template in the T7 system.
•The replisome functions with a looped lagging strand, and both leading- and lagging-strand polymerases are associated with the helicase/primase complex at the fork.
•The T7 replisome functions as a well-coordinated protein machine, with communication between the leading- and lagging-strand polymerases.
•A structural model of the functioning replisome shows the unwinding point of the parental DNA within the leading-strand polymerase, a flexible DNA region that allows looping between the helicase/primase and the lagging-strand polymerase, and a third DNA polymerase that appears to function as a spare to allow polymerase switching on the lagging strand.
•All ssDNA at the replication fork is coated with ssDNA-binding protein, which helps to organize the replisome.
•Discontinuities in the lagging strand are repaired by the action of a specialized T7 nuclease, DNA polymerase, and DNA ligase.
•A transcript synthesized by T7 RNA polymerase is needed to initiate DNA replication, likely via R-loop formation.
Further Reading
Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., & Bourne, P. E. (2000). The Protein Data Bank. Nucleic Acids Res, 28, 235–242.
Cernooka, E., Rumnieks, J., Tars, K., & Kazaks, A. (2017). Structural basis for DNA recognition of a single-stranded DNA-binding protein from Enterobacter phage Enc34. Sci Rep, 7, 15529, doi:10.1038/s41598-017-15774-y
Gao, Y., Cui, Y., Fox, T., Lin, S., Wang, H., de Val, N., Zhou, Z. H., & Yang, W. (2019). Structures and operating principles of the replisome. Science, 363(6429), eaav7003.
Hamdan, S. M., & van Oijen, A. M. (2010). Timing, coordination, and rhythm: Acrobatics at the DNA replication fork. J Biol Chem, 285(25), 18979–18983.
Holt, I. J., & Reyes, A. (2012). Human mitochondrial DNA replication. Cold Spring Harb Perspect Biol, 4(12), a012971.
Kulczyk, A. W., & Richardson, C. C. (2016). The replication system of bacteriophage T7. Enzymes, 39, 89–136.
Lee, J., Chastain, P. D., 2nd, Kusakabe, T., Griffith, J. D., & Richardson, C. C. (1998). Coordinated leading and lagging strand DNA synthesis on a minicircular template. Mol Cell, 1(7), 1001–1010.
Lee, S. J., & Richardson, C. C. (2011). Choreography of bacteriophage T7 DNA replication. Curr Opin Chem Biol, 15(5), 580–586.
Li, Y., Dutta, S., Doublie, S., Bdour, H. M., Taylor, J. S., & Ellenberger, T. (2004). Nucleotide insertion opposite a cis-syn thymine dimer by a replicative DNA polymerase from bacteriophage T7. Nat Struct Mol Biol, 11, 784–790.
Singleton, M. R., Sawaya, M. R., Ellenberger, T., & Wigley, D. B. (2000). Crystal structure of T7 gene 4 ring helicase indicates a mechanism for sequential hydrolysis of nucleotides. Cell, 101, 589–600.
Sinha, N. K., Morris, C. F., & Alberts, B. M. (1980). Efficient in vitro replication of double-stranded DNA templates by a purified T4 bacteriophage replication system. J Biol Chem, 255(9), 4290–4293.
Toth, E. A., Li, Y., Sawaya, M. R., Cheng, Y., & Ellenberger, T. (2003). The crystal structure of the bifunctional primase-helicase of bacteriophage T7. Mol Cell, 12, 1113–1123.
Wallen, J. R., Zhang, H., Weis, C., Cui, W., Foster, B. M., Ho, C. M. W., Hammel, M., Tainer, J. A., Gross, M. L., & Ellenberger, T. (2017). Hybrid methods reveal multiple flexibly linked DNA polymerases within the bacteriophage T7 replisome. Structure, 25, 157–166.
How did they test that?
Are leading- and lagging-strand synthesis coupled?
Lee, Chastain, Kusakabe, Griffith, and Richardson (1998) exploited a powerful “minicircle” assay and the DNA replication system of bacteriophage T7. The minicircle, constructed with chemically synthesized oligonucleotides, consists of a 70-bp circle with a 5′-single-stranded extension (panel A). The 3′ end at the branch point serves as the site for initiation of leading-strand synthesis, while lagging-strand synthesis initiates when primase sites are exposed during the first round of leading-strand synthesis. The substrate is replicated efficiently (panel B), producing duplex products that can exceed 20,000 bp (hundreds of times around the circle). A key feature is that the two strands differ greatly in nucleotide content — the leading-strand product contains a great excess of dG over dC residues, while the lagging-strand product is just the opposite (the only exception is from the primase sites that