Читать книгу Replicating And Repairing The Genome: From Basic Mechanisms To Modern Genetic Technologies - Kenneth N Kreuzer - Страница 20

The two major domains of the T7 helicase/primase protein have distinct functions — one catalyzes the unwinding of the parental DNA duplex (helicase) and the other provides RNA primers for lagging-strand synthesis of Okazaki fragments (primase). As we see in Chapters 3 and 4, cellular replication systems utilize two distinct proteins for these two functions, although even in those cases the two proteins interact functionally and communicate with each other.

Focusing first on DNA unwinding activity, T7 helicase translocates in the 5′ to 3′ direction along a single strand of DNA, unwinding any duplex region that is encountered during this translocation. Recent structural studies support an attractive “hand-over-hand” model for this translocation. Helicase complexes with ssDNA were resolved into multiple hexameric forms that resembled lock-washers rather than symmetrical rings. The image at the top left of Figure 2.4A shows one such hexamer, with the discontinuity of the lock-washer in between the subunits labeled HelA and HelF.³ The key structural transition in the model is that the subunit on the 5′ side of the discontinuity moves up to the 3′ end of the lock-washer, as the discontinuity shifts to the next interface (between HelE and HelF in the first step; Figures 2.4A and B). Given the 5′ to 3′ direction of movement along the DNA, the HelF subunit is essentially moving from the back to the front of the lock-washer. This movement comprises approximately 24 angstroms (10⁻¹⁰ meters) in the 5′ to 3′ direction. Long-range movement of the helicase along the DNA is accomplished by the sequential movement of individual subunits from the back to the front of the lock-washer, as the discontinuity essentially rotates around the ring (Figure 2.4B). Note that the structure at the end of six cycles of subunit movement is identical to the structure at the start, except that the hexamer has advanced along the ssDNA.

Figure 2.4.Movement and loading of the T7 replicative helicase. Recent structural studies strongly support a “hand-over-hand” mechanism for translocation of the T7 helicase along ssDNA (Gao et al., 2019). In panel A, closely related helicase-DNA structures illustrate key features of the mechanism (see text for detailed discussion). Gao et al. (2019) refer to the six subunits as HelA, HelB, HelC, HelD, HelE, and HelF. The lock-washer image between panels A and B illustrates the overall structure, with DNA indicated by the arrow (pointing 5′ to 3′). The sequential movement along ssDNA is illustrated in the cartoon in panel B. The dotted lines represent the inter-subunit contacts and are only shown in this cartoon for the junction across the discontinuity of the lock-washers. The arrows to the right of each cartoon figure are in the same color as the subunit that is moving up the hexamer structure. Notice that the overall hexamer is moving in the 5′ to 3′ direction along the DNA. Panel C depicts a ring-opening model for helicase loading. In solution without DNA, the T7 helicase forms heptamers, but hexamers are observed after loading onto ssDNA. The model proposes that one subunit of the heptamer is ejected as the ring rotates into the hexameric lockwasher form. Panel A is reproduced from Gao et al. (2019), with permission from the American Association for the Advancement of Science; permission conveyed by Copyright Clearance Center, Inc. Panel B was modified from a figure by Gao et al. (2019), and panel C from a figure by Kulczyk and Richardson (2016).

One critical aspect of the model is the role of nucleotide hydrolysis. T7 helicase hydrolyzes dTTP to translocate along DNA. The sites of nucleotide binding are at the interface between adjacent subunits in the hexamer, and given the hexameric state of the protein, there are six potential active sites around the ring (Figure 2.1B, image ii). Each subunit movement across the discontinuity is proposed to be driven by hydrolysis of the dTTP bound to that subunit, which then binds a new dTTP after its repositioning at the front of the lock-washer.

Another critical aspect of the hand-over-hand model is that each subunit has an extension that contacts the adjacent subunit, and this extension reaches across the discontinuity of the lock-washer. The extension is most easily seen in Figure 2.4A images as the red segment (HelF) at the bottom of the blue subunit (HelA), but each subunit contacts its neighbor in this manner. In the cartoon in Figure 2.4B, extension across the discontinuity of the lock-washer is depicted as the dotted line (the other five extensions are not shown in this cartoon for simplicity). The extensions to the adjacent subunits are actually the regions in between the helicase and primase portions of the protein (see Figure 2.1B, image iv; also see Figure 2.1B, image i, where the inter-subunit contacts are very evident).

The ssDNA in these structures was found to be threaded helically through the middle of the hexamer, with each helicase subunit contacting the phosphodiester backbone of two adjacent nucleotides. Six subunits are thus engaged with 12 adjacent nucleotides along the ssDNA, and the subunit that jumps from the back to the front of the lock-washer skips over the 10 intervening nucleotides.

The central hole of the hexameric helicase is not large enough to accommodate duplex DNA, and accordingly any complementary strand that is base-paired in front of the hexamer gets displaced as the enzyme translocates through that region. Translocation along a single strand of DNA is quite fast, greater than 100 nucleotides per second, but the enzyme slows down considerably (more than 10-fold) when it is also stripping a complementary strand from the DNA. This makes sense because DNA base pairs must be disrupted to displace the strand.

The stimulation of DNA polymerase activity by the helicase/primase complex was discussed above. The interaction of these two proteins is mutually beneficial, in that polymerase also stimulates the rate of helicase unwinding by about 10-fold. In a sense, the activity of each enzyme in the absence of the other only hints at its true capabilities. Evidently, these two enzymes form components of a well-coordinated molecular machine, the structure of which will be considered below.

The directionality of a replicative helicase is an important characteristic, because it dictates whether the enzyme is traveling along the leading-strand or the lagging-strand template. In the case of T7 helicase/primase, the 5′ to 3′ directionality places the protein on the lagging-strand template (Figure 2.3). This positions the attached primase on the strand where it needs to synthesize RNA primers for Okazaki fragment synthesis.

DNA helicases must be carefully controlled to prevent accidental unwinding of duplex genomic DNA. We will see in subsequent chapters that cellular replication systems have dedicated helicaseloading proteins, which are regulated to allow unwinding only at the time and place dedicated for genomic replication. Surprisingly, there is no such protein known in the case of phage T7. During in vitro reactions, the T7 helicase can load without any accessory proteins onto ssDNA, even if the DNA is circular. This result implies that either the hexamer is newly assembled around the DNA or that hexamers are somehow loaded through a breach in the ring structure. A hint about the loading mechanism is that T7 helicase/primase readily forms a heptamer in solution without DNA (but a hexamer with ssDNA). One model for loading is that the heptamer loses one subunit as the DNA passes through the resulting void, with the hexamer then closing down around the DNA (Figure 2.4C).

The primase activity of the helicase/primase complex provides tetraribonucleotide primers for the initiation of Okazaki fragments on the lagging strand. Primers are made only at certain template sequences, with primers having the sequences 5′-ACCA-3′, 5′-ACCC-3′, and 5′-ACAC-3′. The catalytic reaction of primases is much like that of standard RNA polymerases. In both cases, the first step is the condensation of two nucleoside triphosphates into a dinucleotide with a 5′ triphosphate end on the first residue, a phosphodiester linkage between the two, and a free hydroxyl on the 3′ position of the second nucleotide residue. Subsequent nucleotide additions follow the paradigm of DNA/RNA polymerase nucleotide addition, with phosphodiester bond formation at the 3′-OH of the most recently incorporated nucleotide residue and release of pyrophosphate.

The interactions between T7 DNA polymerase and the helicase/primase complex facilitate the hand-off of the tetraribonucleotide primer to the DNA polymerase for Okazaki fragment extension. As we will see shortly, the events in lagging-strand synthesis, including primer formation, hand-off, and Okazaki fragment extension, are carefully coordinated in the overall replication process.