Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies. Kenneth N Kreuzer
repair and/or recombination. Obviously, the process of fork regression must be very carefully regulated or else replication would turn into a hopelessly complicated process. Getting back to the DNA damage that caused the problem in the first place, the process of fork regression has driven the damage back into a duplex region, and thereby allows excision repair to occur successfully and fix the problem (Figure 3.6B). Presumably, excision repair is somehow coupled to this process of regression, but the details are not yet clear. Following excision repair, the fork regression must be reversed (Figure 3.6C), and the normal replication fork restarted, and again one assumes that these are all well-coordinated events.
Fork regression can be used to achieve another remarkable outcome, inserting the correct complementary base opposite a damaged base that itself cannot serve as a template for correct base pairing. This can occur in the situation where the leading-strand polymerase has been blocked by a damaged template base, but lagging-strand replication has proceeded a short distance further on. Fork regression can again extrude the two newly synthesized strands into a (mostly) duplex region (Figure 3.7A). In this case, the 3′ end of the new leading strand, which had been blocked by the damage prior to regression, is properly base paired with the new and longer laggingstrand product. Notice that the sequence of the new lagging-strand product would be identical to the sequence of the old leadingstrand template, because it is “the complement of the complement.” Thus, the lagging-strand product can serve as an accurate template for leading-strand synthesis! DNA polymerase extension of the 3′ end of the leading strand, even for a few bases, has the effect of circumventing or bypassing the original damage (Figure 3.7B). When the regression process is reversed, the leading-strand product has now been extended past the damage, with the correct base opposite the damage (Figure 3.7C). The fork can restart and complete the replication of the genome. The damaged base remains, but the cell can complete its round of cell division and now has another cell cycle to properly repair the damage, for example, by excision repair.
Figure 3.6.Replication fork regression allows repair of blocking template lesions. The two newly synthesized strands at the replication fork are complementary to each other and can thereby anneal to each other and allow the fork to “back up” (step A). Once the lesion is restored into a region of duplex DNA, repair pathways described later in this book can repair the damage (step B). Reversal of the regression restores a normal replication fork structure (step C), and the process of replication can then resume.
Figure 3.7.Error-free bypass of a blocking lesion via replication fork regression. After blockage of the leading-strand polymerase, replication fork regression places the blocked 3′ end in a duplex with the lagging-strand product (step A). This allows accurate incorporation of several base residues by DNA polymerase (step B). Reversal of regression (step C) then allows resumption of normal DNA synthesis, with the blocking lesion bypassed.
Importantly, fork regression-driven bypass occurs in an accurate, mutation-free manner. A different and very important form of lesion bypass involves specialized DNA polymerases, and will be covered in Chapter 12. Overall, it is clear that numerous sophisticated pathways have evolved, allowing the completion of bacterial DNA replication even when the DNA is damaged.
3.8Summary of key points
•The circular E. coli chromosome is replicated by two oppositely oriented replisome complexes that each traverse the DNA at a speed of about 1000 base pairs per second.
•The heart of the E. coli replisome is the DNA polymerase III complex, which contains the multi-subunit polymerase, the clamp loader and clamp.
•The sliding clamp encircles the DNA and allows DNA polymerase to synthesize long stretches of DNA without falling off the template (i.e., increasing polymerase processivity).
•The five-subunit clamp loader harnesses ATP binding and hydrolysis to crack open the clamp ring structure and load the clamp onto DNA at primer-template junctions.
•The bacterial helicase is a hexamer that travels along the lagging-strand template, associated and interacting with both the bacterial primase and the leading-strand polymerase.
•The E. coli replisome can continue past the site of lagging-strand damage, leaving a short single-stranded region containing the damaged base.
•E. coli ssDNA-binding protein is a tetramer that protects ssDNA at the replication fork, prevents formation of deleterious secondary structures, and interacts with other proteins in the replisome.
•DNA polymerase I replaces ribonucleotide residues from Okazaki fragments, and DNA ligase seals the remaining nicks.
•E. coli and other bacteria have multiple pathways for restarting replication and otherwise rescuing replisomes that have encountered serious problems such as blocking lesions and breaks.
Further Reading
Atkinson, J., & McGlynn, P. (2009). Replication fork reversal and the maintenance of genome stability. Nucleic Acids Res, 37(11), 3475–3492.
Hedglin, M., Kumar, R., & Benkovic, S. J. (2013). Replication clamps and clamp loaders. Cold Spring Harb Perspect Biol, 5(4), a010165.
Johansson, E., & Dixon, N. (2013). Replicative DNA polymerases. Cold Spring Harb Perspect Biol, 5(6), a012799.
Kelch, B. A., Makino, D. L., O’Donnell, M., & Kuriyan, J. (2012). Clamp loader ATPases and the evolution of DNA replication machinery. BMC Biol, 10, 34, doi:10.1186/1741-7007-10-34.
Kong, X. P., Onrust, R., O’Donnell, M., & Kuriyan, J. (1992). Three-dimensional structure of the β subunit of E. coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell, 69, 425–437.
Langston, L. D., & O’Donnell, M. (2006). DNA replication: Keep moving and don’t mind the gap. Mol Cell, 23(2), 155–160.
O’Donnell, M., Langston, L., & Stillman, B. (2013). Principles and concepts of DNA replication in bacteria, archaea, and eukarya. Cold Spring Harb Perspect Biol, 5(7), a010108.
Stukenberg, P. T., Studwell-Vaughan, P. S., & O’Donnell, M. (1991). Mechanism of the sliding β-clamp of DNA polymerase III holoenzyme. J Biol Chem, 266(17), 11328–11334.
Yao, N. Y., & O’Donnell, M. (2008). Replisome dynamics and use of DNA trombone loops to bypass replication blocks. Mol Biosyst, 4(11), 1075–1084.
Yao, N. Y., & O’Donnell, M. (2012). The RFC clamp loader: Structure and function. Subcell Biochem, 62, 259–279.
Yeeles, J. T., Poli, J., Marians, K. J., & Pasero, P. (2013). Rescuing stalled or damaged replication forks. Cold Spring Harb Perspect Biol, 5(5), a012815.
How did they test that?
Does the sliding clamp encircle DNA?
Stukenberg et al. (1991) analyzed the mechanism by which E. coli sliding clamp stimulates DNA polymerase. One approach was to ask about the nature of clamp binding to DNA. They loaded radioactive clamps onto (nicked) duplex circular DNA, subjected the mixture to various manipulations, and then analyzed the samples with gel-filtration columns. In these columns, large intact protein-DNA complexes elute in the early fractions (around fraction 15), while smaller unbound proteins elute later (fractions 25–35). Previous experiments verified these elution positions. In panel A, radioactive clamp protein is found to