Known and Proposed Components of the DNA replication complex
1. Introduction
Major processes in the cell often involve the coordinated and efficient assembly of macromolecular complexes; such examples include: RNA transcription, DNA replication, translation, and cellular motion. These processes can be likened to miniature forms of machines, so-called “molecular machines” with multiple components and motors at their heart driving the systems. This term has been used by several researchers, which equate many of life’s inner workings as homologous to machines; albeit much more efficient than their macro-type counterparts [104]. In 1998, Bruce Alberts wrote an elegant article for
2. Phases of DNA replication
The replication of DNA during the Synthesis (S) Phase of the cell is generally differentiated into distinct stages. The first is the binding and
The formation of an active pre-replication complex at the origin, and the subsequent formation and activation of the CMG replicative DNA helicase allows for the recruitment of DNA polymerase α primase, which is necessary for the synthesis of RNA primers and a short DNA extension of those primers. Also recruited is RPA, the major ssDNA binding complex necessary to prevent the re-annealing of the DNA duplex [132], and topoisomerase I, which resolves the compression of the DNA helix caused by progression of the replication fork along the DNA duplex (
Following elongation, the RNA primers and the RNA-DNA linkages are removed through the actions of the flap endonuclease-1 (FEN1) nuclease and/or Pif1 helicase and Dna2 nuclease, assisted by RPA and DNA polymerase δ [74, 105, 108]. Following the removal of the primers, gaps are filled in, apparently by the action of the DNA polymerase δ and its cofactors, and the final DNA strands are ligated by DNA ligase I into long uninterrupted DNA chains. The removal of all the primers, filling of the subsequent gaps, and the final ligation of the products represent the completion of S-phase.
3. Model systems for elongation of DNA replication
As mentioned previously, eukaryotic cellular DNA replication is highly complicated, and only recently has the replicative DNA helicase finally been identified as MCM2-7 complexed with Cdc45 and the GINS complex (CMG) [91]; furthermore, the complex nature of assembly and regulation of this CMG replicative helicase has limited the ability to study the eukaryotic replication fork biochemically. However, early mechanistic studies of eukaryotic DNA replication were largely carried out using the small DNA tumor virus SV40 and to a lesser extent the papillomaviruses. What makes these viruses ideal models for the mechanistic study of eukaryotic DNA replication? One reason lies in their small genome size. To facilitate their duplication, these viruses make the most of their small number of ORFs by combining multiple replication functions into one or two proteins, and relying primarily on the host cell DNA replication machinery (see Table 1). In addition, the lack of these viruses utilizing the once-and-only-once per S Phase regulation of DNA replication means that their DNA replication systems were not subject to the complicated and constraining regulatory systems that control replication of cellular DNA. SV40 DNA replication is driven by a single viral protein, SV40 large T-antigen (Tag), a protein that combines all the core DNA replication functions of the cellular initiation and origin activation proteins listed above for eukaryotic DNA replication. Tag recognizes and binds to the SV40 origin of replication, melts the DNA helix surrounding the origin, and establishes itself into a double hexameric structure. Tag then recruits the cellular DNA replication factors: RPA, topoisomerase I, and polymerase α primase. These four replication factors are all that is required for the initiation of SV40 DNA replication through the initial synthesis of RNA-DNA primers. Following these initiation events, the clamp loader, RFC, and the polymerase processivity factor, PCNA, are recruited and loaded, which leads to the binding and activity of DNA polymerase δ, which extends both lagging and leading strands in this viral DNA replication system. As in the mammalian system, Okazaki fragments are processed by FEN1, DNA helicase 2, and DNA ligase 1, completing synthesis of the viral DNA genomes. It was the early studies of this viral DNA synthesis system that elucidated these basic mechanisms of how eukaryotic DNA replication is carried out.
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Origin Recognition/Initiator | Orc complex (2-6) | T-antigen (Tag) | E2/E1 |
pre-RC | Cdc6, Cdt1, Cdc45, Geminin, MCM10, Sld2(RecQL4), Sld3, Dpb11(TopBP1) | Tag | E2/E1 |
Helicase | MCM 2-7, GINS, Cdc45 | Tag | E1 |
SSB | RPA | RPA | RPA |
Torsional relaxation | Topoisomerase I | Topoisomerase I | Topoisomerase I |
Clamp loader | RFC | RFC | RFC |
Processivity factor | PCNA | PCNA | PCNA |
DNA polymerases | DNA pol α primase, DNA pol δ, DNA pol ε | DNA pol α primase, DNA pol δ | DNA pol α primase, DNA pol δ |
Accessory factors | Mrc1(Claspin) | None | None? |
Similar findings were also found for another virus family, the papillomaviruses, which have also proven to be an apt model for cellular DNA replication mechanisms due to their dependence on the host replication machinery. Initial studies were carried out in the bovine version BPV-1, and later corroborated with several human HPV isotypes. In general, papillomaviruses follow the same mode and progression of events found in SV40, except for the need for two viral proteins instead of the single Tag protein required for SV40. In addition, PV appears to require other cellular factors that SV40 does not [73, 80, 87], which to date remain unidentified. In papillomavirus DNA replication, the E2 protein assists and directs faithful viral origin recognition of E1 [79, 90, 110, 126], while E1 itself serves the role of the replicative DNA helicase, melting the DNA around the origin of replication and establishing itself as a double hexameric helicase. In a fashion similar to that of SV40 Tag, E1 also acts to recruit the cellular DNA replication proteins to the PV DNA replication fork [36, 113, 131]. E1 itself is a weak origin binding protein, but can bind to and unwind DNA even in the absence of E2 at high E1 concentrations, even on DNA without an apparent E1 binding sequence and is therefore relatively nonspecific without E2 [66]. Furthermore, following establishment of the double hexamer, the E2 protein is purportedly absent from subsequent steps of DNA replication, indicating E1 is the only viral protein implicated in the actual HPV elongating DNA fork [72]. Otherwise, these two small DNA viruses display very similar mechanisms of replication, especially during the elongation phase. So why rely on two very similar viruses as models and not just SV40? One reason is that by comparing and contrasting the DNA replication mechanisms in two subtly different systems, one gains further insight into the mechanisms of DNA replication. In specific aspects of DNA replication, one or the other virus might provide a more apt reflection of the mechanism of cellular DNA replication. Another reason lies in the diseases each virus causes and the implications for antiviral research. Although SV40 Tag is a potent transforming agent for cell culture due to its ability to inactivate p53, Rb protein, and many other components of the cell, SV40 itself does not appear to readily cause tumors in humans. Conversely, human papillomaviruses are the major cause of cervical, anogenital, and oral cancers and represent the major cause of infectious-agent-induced cancers in humans. These viruses represent historically important and still valuable models for DNA replication and can still be used to elucidate hitherto unknown mechanisms of mammalian DNA replication. Furthermore, the replicative DNA helicases of these viral DNA replication systems still provide the best biochemical system for investigating the role of DNA helicases in the elongation stage of eukaryotic DNA replication.
4. Replicative DNA helicases
When the structure of the DNA double helix was first proposed, one of the major questions concerning the replication of dsDNA was how the duplex would be opened to facilitate reading of the base sequence encoded by the DNA. The first such discovered protein that could carry out this function was the prokaryotic helicase of
Various models have been proposed for how DNA helicases unwind the DNA helix. Some early proposals included the monomers binding to the DNA backbone and essentially rolling one DNA strand away from the other using the circular nature of the hexamer. Other models included a hexamer ‘embracing’ ssDNA, excluding it from its partner, or two hexamers acting at a distance pumping dsDNA through their central pore. Some studies indicate the double hexamers stay associated during elongation, and this led to a double hexameric DNA pumping mechanism that pumps dsDNA through the central pore somehow splitting the helix [42]. The more recent structural studies of the BPV1 E1 helicase bound to DNA, ATP, and ADP indicate an intricate hybrid model whereby the E1 hexamer pumps ssDNA through each central pore in a staircase type mechanism as ATP is bound and hydrolyzed by each subsequent E1 monomer [32, 33, 109]. In this model E1 uses the ATP binding/hydrolysis-induced conformational changes of the individual monomers to drive each nucleotide base of the enclosed ssDNA template through the central pore, displacing the hybridized (lagging-strand template) DNA strand freeing it to be available as a template for lagging strand DNA synthesis [32]. Although the model for helicase action based on the SV40 Tag structure was not the same, the Tag structure was done in the absence of ssDNA, and the structural information on the Tag hexamer would be consistent with a helicase model similar to that of E1.
5. Helicase interactions with replication proteins that initiate elongation
As stated previously, DNA replication proteins commonly recruited by both of these viral replicative helicases are: RPA, topoisomerase I, and DNA polymerase α primase. In this section, we will look closer at the individual and combinatorial interactions between the helicase and these necessary DNA replication factors that are intimately involved in both the initiation and elongation stages of DNA replication. In many cases, studies have focused on specific interactions, often detailed down to specific amino acid residues required for recruitment of these factors. Various groups have used the powerful ability to investigate the interactions of these factors with the viral helicases both
6. Helicase interactions with replication proteins that initiate elongation: Topoisomerase I
The unwinding action of the DNA replication fork driving along the DNA helix creates torsional stress and overwound DNA that must be relieved to allow replication to proceed. Topoisomerases are enzymes that help relieve this stress and aid in maintaining chromosome structure and integrity by modifying DNA topology, and resolving specific DNA structures that arise from cellular processes such as DNA repair, replication, transcription, recombination and chromosome compaction [13]. These processes result in compression (positive supercoiling) of the DNA helix and the entanglement of DNA segments and chromosomal regions that can lead to cytotoxic or mutagenic breaks in the DNA if left unmanaged [127]. Hence, topoisomerases play a vital role in living cells, particularly during DNA replication.
Enzymatically, topoisomerases act through the action of a nucleophilic tyrosine; the enzyme cleaves one or more DNA strands and generates an enzyme-DNA complex that serves to prevent the release of nicked or broken DNA that could possibly result in chromosome damage [127]. After passage of one or more DNA strands through this transient break(s), the topoisomerase re-ligates the strands leaving the original DNA sequence intact. Though all topoisomerases have this feature in common, topoisomerases are separated into two classes, type I and type II, depending on whether they cleave one or two strands of DNA, respectively [127]. Type I topoisomerases act on one strand, and generally pass a single DNA strand through the transient break, while type II topoisomerases break both DNA strands and generally pass dsDNA through the transient break. Type I topoisomerases generally work in front of replication or transcription forks, to relax positive supercoils in a highly processive manner; while type II topoisomerases are involved in untangling intertwined duplex DNA such as that found in newly replicated molecules or during chromosome resolution during cell division [30].
Topoisomerases have roles in each of the major replicative phases: initiation, fork progression and termination. During DNA replication in eukaryotes, topoisomerases have been observed to bind directly to the replication origin to aid in activation in the initiation phase [45, 127]. During strand synthesis, topoisomerases are required to alleviate compression of the DNA helix caused by positive supercoiling that results from DNA unwinding, which is mediated by replicative helicases [127]. Topoisomerases are also required for daughter strand resolution. Eukaryotes rely on topoisomerase I (topo I) to fulfill the initiation and elongation functions during DNA replication [127].
Human topo I is an ATP-independent, 100-kDa monomeric protein capable of relaxing positive or negative superhelical twists by making a transient single-strand break, thus relieving the tension generated by the replicative helicases during the DNA-unwinding process [61, 127, 135]. Topo I can be divided into four domains: the highly charged NH2 –terminal domain; the conserved core domain; a short, positively charged linker domain, which links the N-terminal domain to the core domain; and the highly conserved COOH-terminal domain, which contains the active-site tyrosine [116]. Due to the topologically constrained nature of a circular dsDNA molecule, it is no surprise that topo I is required for the replication of the genomes of small circular double-stranded DNA viruses. The role of topo I in DNA replication of the small DNA circular DNA viruses was first noted when it was observed that the extent of DNA replication in SV40 DNA replication
While the role of topoisomerases in DNA replication had always been presumed to be due to their need to resolve topological constraint, more recent studies have indicated that topo I plays additional, highly specific, roles in DNA replication of the small DNA viruses, SV40 and PV. Topo I appears to be involved in the very earliest stages of DNA replication, namely origin recognition. It is evident that topo I is stably associated with the initiation complex and is one of the first cellular proteins to be recruited to the initiation machinery [11,45]. Topo I was shown to preferentially associate with the fully formed Tag double hexamer initiation complexes and to be recruited to the initiation complex prior to the beginning of unwinding [11]. This stable association of topo I with Tag results in an increased specificity of Tag for duplex unwinding at the origin by inhibiting unwinding at non-origin sites [39]. Perhaps for this reason, topo I was observed to be required at initiation to stimulate DNA replication
Topoisomerases have been proposed to act together with DNA helicases as “swivelases”, tightly coordinating DNA duplex unwinding with the topoisomerase relaxing activity during DNA replication [15, 30, 61]. With the progression of the replication fork and unwinding of duplex DNA, topo I is needed to release the torsion created by the progressing replication fork [37]. Optimally topo I should be present and its activity regulated to suit the pace of the helicase [37]. This suggested that there might be direct interactions between the helicases and topo I, and that might be modulation of function due to these interactions. The early finding that topo I was localized at SV40 DNA replication forks supported this concept [4], as did evidence that topo I played an important role in the elongation phase of SV40 DNA replication. Reports of the interactions between SV40 TAg and E1 with topo I were also consistent with the swivelase model [15, 133]. The demonstration that E1 stimulates the enzymatic activity of topo I up to seven-fold and that SV40 TAg also stimulates topo I activity (R. Clower and T. Melendy, unpublished results) provided the first evidence of the cooperative nature of this interaction predicted by the swivelase model [15]. Based on these studies it is clear that the viral helicases interact productively with topo I at DNA replication forks forming active coordinated swivelase molecular machines.
The physical interactions between the viral helicases and topo I have been investigated. In 1996, it was found that two independent regions of Tag, one N-terminal and one C-terminal, bind to the cap region of topo I (see Fig. 1), and binding can take place while DNA-bound. Similarly, for PV E1 it was also observed that topo I binds two distinct regions within E1, within E1’s DNA binding domain (DBD) and at the C-terminus [15, 45]. The E1 C-terminal region was shown to enhance topo I relaxation activity, and to a lesser extent, so did a truncation that included the DBD with additional sequence, flanking either side of the DBD [15]. More detailed studies identified mutants in the DNA binding domain of Tag that were unable to unwind the DNA and were partially defective in their association with topo I, suggesting that this interaction maybe important for proper unwinding of viral DNA at replication forks [114]. More recently, four specific amino acid residues within the C-terminal domain of Tag when mutated were shown to exhibit decreased topo I binding and to abolish SV40 DNA replication
7. Helicase interactions with replication proteins that initiate elongation: Replication protein A
One of the first proteins identified as necessary for eukaryotic DNA replication is arguably also one of the most important DNA binding proteins in the cell, the ssDNA binding complex, Replication Protein A (RPA). RPA is a heterotrimeric complex conserved in all eukaryotes, and also shows strong homology to the ssDNA binding proteins of archaebacteria [57, 59]. The human RPA complex is comprised of three subunits, RPA70, RPA32, and RPA14, and the complex binds to ssDNA with extremely high affinity (approximately 10-9 to 10-10 M [62]), showing much lower affinity for dsDNA. RPA binds ssDNA with a distinct polarity, in a 5’->3’ orientation [22, 51]. Like SSB [132], RPA is required for DNA replication
RPA exhibits several DNA binding states. RPA70 has three ssDNA binding sites or oligonucleotide binding (OB) domains and RPA32 has one OB domain [8, 121]. When only RPA70 interacts, this is a lower affinity compacted state, binding to only 8-10 nts. When all four OB domains bind, this represents a higher affinity extended mode that spans ~30 nts [7]. The ability of other proteins to facilitate these binding modes in turn impact the binding of RPA to ssDNA, either covering or exposing various stretches of ssDNA. Since several other proteins bind to RPA through its OB domains, this facilitates a model in which RPA cooperatively hands off and orients the binding of each DNA replication protein through increasing affinity with the subsequent factor [64, 89, 138].
7.1. RPA loading onto ssDNA by replicative DNA helicases
RPA plays many roles in the initial steps of elongation as well as throughout DNA replication. Due to its role in ssDNA stabilization, RPA is one of first proteins required following the unwinding of dsDNA. The critical question here is how this process is coordinated in relation to the double hexameric helicase. The RPA heterotrimer itself makes direct contact with the helicase, be it MCM, SV40 Tag, or PV E1 [3, 43, 77, 95, 101, 130]. The first such studied interaction was through Tag, which interacts with RPA through the helicase’s origin binding domain (OBD) (Figure 1). The importance of this interaction is implied by the absolute necessity for RPA for SV40 replication, RPA cannot be replaced by ssDNA binding proteins from
Evaluation of the multiple interactions between RPA, E1 and ssDNA in various combinations led to development of a novel model for how DNA helicases may ‘load’ ssDNA binding proteins onto ssDNA being displaced through helicase action [77]. RPA binds well to the E1 protein, but only in the absence of free ssDNA. When RPA was prebound to short (~10 nt) stretches of ssDNA, thereby adopting the short compacted form of RPA, it still bound to E1 as well as RPA not bound to DNA. However, when RPA was bound to longer ssDNA templates (~30 nt or longer), consistent with RPA being in its fully-engaged extended form, RPA would no longer bind to E1. This implied a ‘releasing mechanism’ by which the E1-RPA interaction would be released upon RPA binding to ssDNA in RPA’s extended form. Based on this data, a model was developed in which free, non-ssDNA-bound RPA is bound by E1. As the E1 helicase unwinds the dsDNA, producing ssDNA, it positions the RPA to bind to the newly exposed ssDNA, releasing RPA from the helicase complex (see Figure 2). As the helicase progresses, subsequent helicase monomers bring subsequent RPA molecules to the ssDNA continuously displaced by helicase action [77]. Very similar results were later shown for SV40 T-antigen, leading to a nearly identical model for RPA placement onto ssDNA during SV40 T-antigen helicase progression [9, 54]. Of course, this simplified model does not take into account topo I or polymerase α primase interactions, but it does suggest how the newly produced ssDNA can be rapidly coated with RPA to prevent reannealing or hairpin formation, and to protect from nuclease attack.
8. Helicase interactions with proteins that initiate elongation: DNA polymerase alpha-primase
In bacteria and the T4 bacteriophage, the importance of the primase is clear as they are linked physically to the helicase, which is necessary for efficient lagging strand synthesis [19, 92]. In the T7 bacteriophage, this is even more evident as the primase is actually fused to the functional hexameric helicase [31, 99, 102]. In a more complex fashion, in the mammalian system, GINS/ctf4 are required to link the helicase to the catalytic core of DNA polymerase (pol) alpha [40, 120, 140]. Clearly the interaction between primase and the helicase machinery is conserved throughout evolution.
Pol α primase was the first eukaryotic polymerase discovered in 1957 and was thought to be the only replicative DNA polymerase. The later discovery of the proofreading and highly processive polymerases δ and ε indicated that this was not the case [49, 50]. Pol α primase is a heterotetrameric complex comprised of a large p180 catalytic subunit, the regulatory p68 “B” subunit, and the two primase subunits of p55 and p49. Pol α primase is critical for first synthesizing an approximately 10 nt RNA primer, followed by a short ~20-30 nt DNA extension [23, 41, 119]. Polymerase switching then occurs on this RNA/DNA primer through the action of the eukaryotic clamp-loading complex, RFC, which loads the eukaryotic sliding clamp, PCNA, and then a processive DNA polymerase (DNA pol δ or ε) for synthesis of both leading and lagging DNA strands [124, 128]. RFC is integral here, as it competes with RPA for the end of the primer, disrupting the RPA-pol α interaction and allowing polymerase switching [138]. As with many of the core aspects of eukaryotic DNA replication, the functions of pol α primase were largely elucidated using the SV40 system. Pol α primase is absolutely essential for SV40 DNA replication
The interactions between E1 and pol α primase show some differences between those found with SV40 Tag. Early studies indicated that the p180 catalytic subunit interacted with the N-terminal half of E1, while the p68 subunit interacted with the C-terminus of the helicase [18, 83]. A later study then looked closely at the role of the E1 interaction with pol α primase in regards to supporting HPV-11 DNA replication
9. Interactions between replication proteins that initiate elongation: coordination
While the earlier sections have alluded to interplay between the multiple cellular replication factors that interact with the viral helicases during DNA replication, the complexity of the interplay between these interactions is what truly epitomizes the term Molecular Machines.
9.1. RPA’s involvement in de-repression of priming
While the interaction of the viral helicases with RPA has been shown to have a direct effect, apparently through the placement of RPA on the ssDNA being displaced by helicase action, this interaction has also been shown to play another vital role in DNA replication: de-repression of priming. RPA binds directly to pol α primase [10, 28, 96], and can stimulate the fidelity and processivity of pol α primase activity [10, 81]. However, when RPA is present in excess, which it is in human cell nuclei [76], RPA strongly represses synthesis of primers by pol α-primase, likely due to the high affinity of RPA out-competing pol α primase for the ssDNA template [16, 88]. While Tag and pol α primase are required for correct initiation of SV40 DNA replication [27, 130], and the interaction between Tag and pol α-primase is sufficient for stimulation of RNA/DNA primer synthesis by pol α-primase on ssDNA [16], these are insufficient for efficient primer synthesis when there is competition with ssDNA binding proteins. Tag can de-repress primer synthesis by pol α-primase, but only when the ssDNA template is coated by RPA, and not by other ssDNA binding proteins or evolutionarily divergent RPAs [88]. The interaction between Tag and RPA is vital for de-repression of priming [88, 111]. E1 has similarly been shown to interact with RPA, and RPA is required for PV DNA replication (and RPA cannot be replaced by other ssDNA binding proteins in PV DNA replication). So while the E1-RPA interaction has not been shown to be essential for priming de-repression during PV DNA replication, this is nonetheless likely to be the case.
9.2. Topo I’s involvement in priming
Similarly, in addition to its roles in origin recognition/specificity and release of DNA helix compression during elongation, another role for topo I was elucidated when it was observed that topo I induces pol α-primase to synthesize larger amounts of primers with higher molecular weight [60]. In this study, Tag mutants that failed to bind topo I normally did not participate in the synthesis of expected amounts of primers or large molecular weight DNA molecules, indicating that the association of topo I with the C-terminal Tag binding site is required for these processes. Whether this is due to a direct effect on Tag function at the replication fork, or due to an indirect effect on pol α-primase through Tag (analogous to the effect of the RPA-Tag effect on priming by pol α-primase described above) is unclear. Additionally, topo I was shown to bind directly to RPA, and RPA binds directly to pol α-primase, and can stimulate its DNA polymerase activity. It is unclear whether or not RPA may be influencing the interaction of Topo I with pol α-primase, or vice versa [60]. However these interactions are integrated, the binding of topo I to the helicase domain of Tag significantly enhances the synthesis of DNA-RNA primers and their extension by pol α-primase.
9.3. Helicase interactions with other proteins involved in elongation
What of helicase interaction with the other proteins involved in DNA replication elongation? In the model systems of SV40 and PV little has been elucidated about any direct interactions. Of the proteins involved in elongation, very little is known about the role of helicase interaction with pol δ, RFC, PCNA, or the proteins involved in primer removal: RNaseH, DNA2, Fen I, or DNA ligase I. In the accepted model of SV40 DNA replication, the first primers synthesized by pol a primase on the two strands at the origin become the primers for the leading strand of the opposite fork [124]. After recruitment of RFC, PCNA and pol δ, the leading strand polymerase continuously tracks along behind the helicase action. Since the helicase, in this case Tag, unwinds dsDNA at the relatively slow rate of approximately 200 bp/min [93] while pol δ/RFC/PCNA polymerizes at about 80 nts/sec [12], it is reasonable to speculate that the slower speed of the helicase limits the polymerase in such a way to coordinate the entire machinery mechanism. However, the speeds of polymerases are often assayed on artificial templates, and this rate for pol δ/RFC/PCNA is faster than the measured rate of eukaryotic replication forks (~ 2 kb/min). Conversely the measured speed of Tag is far slower than the measured rate for eukaryotic replication forks. It is likely that coordination between the various factors and complexes involved in the replication fork lead to the final replication fork rate that is not dependent on any one factor, but is a characteristic of the coordinated complex. Indeed, it is critical that these machines are tightly regulated; without a tight molecular machine at the fork, there would be wild exposure of ssDNA via the helicase leading to DNA damage signaling. It should also be noted here that DNA pol ε is not needed in SV40 DNA replication [141]. This finding may be due to the lack of a need for two replicative helicases to duplicate small virus genomes. Alternately, DNA pol ε and TopBP1 (Dbp11) play roles in initiation in mammalian replication; this role may be dispensable or even interfere with the Tag/E1 initiator functions [82, 84]. In
9.4. Extrapolation to the cellular chromosomal replication fork
The cellular ‘replicative helicase’ is still poorly defined. Some have designated the human CMG helicase (a large 11 subunit complex comprising Cdc45 and the MCMs and GINS sub-complexes [91]) to be the replicative helicase, while others have designated the RPC, the “replisome progression complex”, comprised of the CMG in complex with Mrc1 (Claspin), Tof1 (Tim or Timeless), Csm3 (Swi3/Tipin), Ctf4 (And-1), and the FACT heterodimer (Spt16, and Pob3 (SSRP1) as the ‘true replicative helicase’ [39]. This study found that MCM10 and topo I associate weakly with this RPC complex, although it is unclear with which specific subunit. It is unknown if the MCM helicase itself interacts with topo I; however, considering the elaborate number of regulatory subunits now known in the eukaryotic helicase supercomplex, this may not be necessary, and may be unlikely. The GINS complex of CMG can bind to and directly stimulate the activity of pol α-primase [21]. A later study showed that the Ctf4 subunit couples the MCMs to pol α-primase and the Mrc1 subunit interacts with polymerase ε [40]. Other studies have found that both Mcm10 and Cdc45 interact with pol α-primase and also found that loss of Mcm10 in yeast led to uncoupling of the MCMs from pol α-primase and resulted in large stretches of ssDNA, a potent DNA damage signal [67, 107]. In human cells, Mcm10 has been suggested to interact with and regulate pol α-primase levels and prevent inappropriate induction of DNA damage [14]. RPA interacts with many components of the RPC, including Mcm3-7, Cdc45, and Claspin (Mrc1) and requires Mcm for chromatin localization [95]. It is intriguing that only RPA appears to directly interact with the Mcms in eukaryotes; this may be due to the intimate linkage with ssDNA and the helicase machine and the highest priority of multicellular organisms to prevent the aberrant signaling of DNA damage through ssDNA coating by RPA. Additionally, in the absence of the RPC interacting protein Mcm10 or in the presence of a mutant zinc finger bearing Mcm10, RPA is also prevented from loading [55]. In general, the major components of the elongation machinery interact with the replicative helicase in eukaryotes through multiple layers of regulation as the RPC complex, a feature that is nonexistent in the simplified machinery presented by these small DNA viral systems. These viral factories simplify the entire complex by using their own central multifunctional helicases. But this simplification has led to the ability to use these viral systems as models where the biochemical nature and functions of these important interactions that occur at the interface of initiation and elongation can be studied.
10. Conclusion
Replicative DNA helicases, modeled by the SV40 and PV DNA replication systems, play complex roles coordinating the multiple actions of multiple DNA replication factors at eukaryotic replication forks. Their interactions with topo I are involved in origin recognition/specificity, DNA helix decompression function, and primer synthesis. Their interactions with pol α-primase are vital for primer synthesis. Their interactions with RPA are involved in loading of RPA onto ssDNA, and de-repression of priming on RPA-coated ssDNA. And the complex interplay between all these factors is intricate, highly-regulated, and appears to be coordinated at least in large part, through the action of the replicative helicases.
Using this wealth of knowledge about the viral replication forks, we have assembled a likely model of replication elongation using the viral helicases as the central molecular machine at the fork. For ease of the various steps of elongation, only a single helicase is pictured in this model (Figure 3). Following assembly of the replication machinery at the viral origin, there is a very intricate four-way interaction comprised of the helicase, topo I, RPA and pol α primase. Topo I has two interactions with helicase; one within the N-terminal half of the helicase and one within the C-terminus. Through these interactions the topo I-helicase interaction assists in helicase origin recognition and creates the swivelase; a machine that couples the unwinding of the DNA duplex with relaxation of torsional stress. During elongation, topo I is likely in front of the helicase to facilitate the easing of positive supercoiling, likely through interactions with the helicase N-terminus. The helicase encircles the leading strand of the newly unwound DNA, actively pumping the leading strand template through the central channel of the helicase and away from the lagging strand replication machinery. While the leading strand template is bound to the central channel and the helicase domain, the lagging strand template is therefore left relatively unprotected. To facilitate a protective role at this point, the OBD of the helicase binds to free RPA, which swings into place as the helicase turns, actively loading RPA onto the lagging strand template. This serves in the role of nuclease protection, as well as preventing aberrant ssDNA structures. However, this coating of the lagging strand template is counterproductive to the process of priming. Therefore, at regular intervals roughly equivalent to the length an Okazaki fragment, the helicase interacts with pol α primase and RPA to facilitate the placement of the pol α primase onto the template, possibly while simultaneously removing RPA in a localized fashion, so that pol α primase can synthesize the RNA-DNA primer. It is intriguing to speculate that it is through this regular placement that Okazaki fragments are placed and spaced; primarily through helicase action and its protein-protein interactions with the primase. Although given the size of eukaryotic Okazaki fragments, it is likely that interactions with histones may play a role as well. The coordinated and highly regulated roles of the multi-subunit DNA helicase in modulating the proteins and their protein-protein interactions involved in the late initiation and elongation stages of DNA replication clearly play a central organizing role in the molecular machine that is the eukaryotic DNA replication fork.
References
- 1.
Alberts, B. 1998. The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell 92:291-294. - 2.
Amin, A. A., S. Titolo, A. Pelletier, D. Fink, M. G. Cordingley, and J. Archambault. 2000. Identification of domains of the HPV11 E1 protein required for DNA replication in vitro. Virology 272:137-150. - 3.
Arunkumar, A. I., V. Klimovich, X. Jiang, R. D. Ott, L. Mizoue, E. Fanning, and W. J. Chazin. 2005. Insights into hRPA32 C-terminal domain--mediated assembly of the simian virus 40 replisome. Nature Structural & Molecular Biology 12:332-339. - 4.
Avemann, K., R. Knippers, T. Koller, and J. M. Sogo. 1988. Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks. Molecular and Cellular Biology 8:3026-3034. - 5.
Bell, S. P., and A. Dutta. 2002. DNA replication in eukaryotic cells. Annual Review of Biochemistry 71:333-374. - 6.
Bjergbaek, L., J. A. Cobb, M. Tsai-Pflugfelder, and S. M. Gasser. 2005. Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance. Embo J 24:405-417. - 7.
Blackwell, L. J., J. A. Borowiec, and I. A. Mastrangelo. 1996. Single-stranded-DNA binding alters human replication protein A structure and facilitates interaction with DNA-dependent protein kinase. Molecular and Cellular Biology 16:4798-4807. - 8.
Bochkarev, A., and E. Bochkareva. 2004. From RPA to BRCA2: lessons from single-stranded DNA binding by the OB-fold. Curr Opin Struct Biol 14:36-42. - 9.
Bochkareva, E., D. Martynowski, A. Seitova, and A. Bochkarev. 2006. Structure of the origin-binding domain of simian virus 40 large T antigen bound to DNA. Embo J 25:5961-5969. - 10.
Braun, K. A., Y. Lao, Z. He, C. J. Ingles, and M. S. Wold. 1997. Role of protein-protein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase alpha by multiple mechanisms. Biochemistry 36:8443-8454. - 11.
Brill, S. J., and B. Stillman. 1989. Yeast replication factor-A functions in the unwinding of the SV40 origin of DNA replication. Nature 342:92-95. - 12.
Burgers, P. M., and K. J. Gerik. 1998. Structure and processivity of two forms of Saccharomyces cerevisiae DNA polymerase delta. The Journal of Biological Chemistry 273:19756-19762. - 13.
Champoux, J. J. 2001. DNA topoisomerases: structure, function, and mechanism. Annual Review of Biochemistry 70:369-413. - 14.
Chattopadhyay, S., and A. K. Bielinsky. 2007. Human Mcm10 regulates the catalytic subunit of DNA polymerase-alpha and prevents DNA damage during replication. Molecular Biology of the Cell 18:4085-4095. - 15.
Clower, R. V., J. C. Fisk, and T. Melendy. 2006. Papillomavirus E1 protein binds to and stimulates human topoisomerase I. Journal of Virology 80:1584-1587. - 16.
Collins, K. L., and T. J. Kelly. 1991. Effects of T antigen and replication protein A on the initiation of DNA synthesis by DNA polymerase alpha-primase. Molecular and Cellular Biology 11:2108-2115. - 17.
Collins, K. L., A. A. Russo, B. Y. Tseng, and T. J. Kelly. 1993. The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication. Embo J 12:4555-4566. - 18.
Conger, K. L., J. S. Liu, S. R. Kuo, L. T. Chow, and T. S. Wang. 1999. Human papillomavirus DNA replication. Interactions between the viral E1 protein and two subunits of human dna polymerase alpha/primase. The Journal of Biological Chemistry 274:2696-2705. - 19.
Corn, J. E., and J. M. Berger. 2006. Regulation of bacterial priming and daughter strand synthesis through helicase-primase interactions. Nucleic Acids Research 34:4082-4088. - 20.
Davey, M. J., D. Jeruzalmi, J. Kuriyan, and M. O'Donnell. 2002. Motors and switches: AAA+ machines within the replisome. Nat Rev Mol Cell Biol 3:826-835. - 21.
De Falco, M., E. Ferrari, M. De Felice, M. Rossi, U. Hubscher, and F. M. Pisani. 2007. The human GINS complex binds to and specifically stimulates human DNA polymerase alpha-primase. EMBO Reports 8:99-103. - 22.
de Laat, W. L., E. Appeldoorn, K. Sugasawa, E. Weterings, N. G. Jaspers, and J. H. Hoeijmakers. 1998. DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair. Genes & Development 12:2598-2609. - 23.
Denis, D., and P. A. Bullock. 1993. Primer-DNA formation during simian virus 40 DNA replication in vitro. Molecular and Cellular Biology 13:2882-2890. - 24.
Diffley, J. F., and K. Labib. 2002. The chromosome replication cycle. Journal of Cell Science 115:869-872. - 25.
Dodson, G. E., Y. Shi, and R. S. Tibbetts. 2004. DNA replication defects, spontaneous DNA damage, and ATM-dependent checkpoint activation in replication protein A-deficient cells. The Journal of Biological Chemistry 279:34010-34014. - 26.
Donovan, S., J. Harwood, L. S. Drury, and J. F. Diffley. 1997. Cdc6p-dependent loading of Mcm proteins onto pre-replicative chromatin in budding yeast. Proceedings of the National Academy of Sciences of the United States of America 94:5611-5616. - 27.
Dornreiter, I., W. C. Copeland, and T. S. Wang. 1993. Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen. Molecular and Cellular Biology 13:809-820. - 28.
Dornreiter, I., L. F. Erdile, I. U. Gilbert, D. von Winkler, T. J. Kelly, and E. Fanning. 1992. Interaction of DNA polymerase alpha-primase with cellular replication protein A and SV40 T antigen. Embo J 11:769-776. - 29.
Dornreiter, I., A. Hoss, A. K. Arthur, and E. Fanning. 1990. SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha. Embo J 9:3329-3336. - 30.
Duguet, M. 1997. When helicase and topoisomerase meet! Journal of Cell Science 110 ( Pt 12):1345-1350. - 31.
Egelman, E. H., X. Yu, R. Wild, M. M. Hingorani, and S. S. Patel. 1995. Bacteriophage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric helicases. Proceedings of the National Academy of Sciences of the United States of America 92:3869-3873. - 32.
Enemark, E. J., and L. Joshua-Tor. 2006. Mechanism of DNA translocation in a replicative hexameric helicase. Nature 442:270-275. - 33.
Enemark, E. J., and L. Joshua-Tor. 2008. On helicases and other motor proteins. Curr Opin Struct Biol 18:243-257. - 34.
Evrin, C., P. Clarke, J. Zech, R. Lurz, J. Sun, S. Uhle, H. Li, B. Stillman, and C. Speck. 2009. A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication. Proceedings of the National Academy of Sciences of the United States of America 106:20240-20245. - 35.
Fanning, E., V. Klimovich, and A. R. Nager. 2006. A dynamic model for replication protein A (RPA) function in DNA processing pathways. Nucleic Acids Research 34:4126-4137. - 36.
Fouts, E. T., X. Yu, E. H. Egelman, and M. R. Botchan. 1999. Biochemical and electron microscopic image analysis of the hexameric E1 helicase. The Journal of Biological Chemistry 274:4447-4458. - 37.
Gai, D., R. Roy, C. Wu, and D. T. Simmons. 2000. Topoisomerase I associates specifically with simian virus 40 large-T-antigen double hexamer-origin complexes. Journal of Virology 74:5224-5232. - 38.
Gai, D., R. Zhao, D. Li, C. V. Finkielstein, and X. S. Chen. 2004. Mechanisms of conformational change for a replicative hexameric helicase of SV40 large tumor antigen. Cell 119:47-60. - 39.
Gambus, A., R. C. Jones, A. Sanchez-Diaz, M. Kanemaki, F. van Deursen, R. D. Edmondson, and K. Labib. 2006. GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nat Cell Biol 8:358-366. - 40.
Gambus, A., F. van Deursen, D. Polychronopoulos, M. Foltman, R. C. Jones, R. D. Edmondson, A. Calzada, and K. Labib. 2009. A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase alpha within the eukaryotic replisome. Embo J 28:2992-3004. - 41.
Garg, P., and P. M. Burgers. 2005. DNA polymerases that propagate the eukaryotic DNA replication fork. Crit Rev Biochem Mol Biol 40:115-128. - 42.
Gomez-Lorenzo, M. G., M. Valle, J. Frank, C. Gruss, C. O. Sorzano, X. S. Chen, L. E. Donate, and J. M. Carazo. 2003. Large T antigen on the simian virus 40 origin of replication: a 3D snapshot prior to DNA replication. Embo J 22:6205-6213. - 43.
Han, Y., Y. M. Loo, K. T. Militello, and T. Melendy. 1999. Interactions of the papovavirus DNA replication initiator proteins, bovine papillomavirus type 1 E1 and simian virus 40 large T antigen, with human replication protein A. Journal of Virology 73:4899-4907. - 44.
Hodgson, B., A. Calzada, and K. Labib. 2007. Mrc1 and Tof1 regulate DNA replication forks in different ways during normal S phase. Molecular Biology of the Cell 18:3894-3902. - 45.
Hu, Y., R. V. Clower, and T. Melendy. 2006. Cellular topoisomerase I modulates origin binding by bovine papillomavirus type 1 E1. Journal of Virology 80:4363-4371. - 46.
Huang, H., B. E. Weiner, H. Zhang, B. E. Fuller, Y. Gao, B. M. Wile, K. Zhao, D. R. Arnett, W. J. Chazin, and E. Fanning. 2010. Structure of a DNA polymerase alpha-primase domain that docks on the SV40 helicase and activates the viral primosome. The Journal of Biological Chemistry 285:17112-17122. - 47.
Huang, H., K. Zhao, D. R. Arnett, and E. Fanning. 2010. A specific docking site for DNA polymerase {alpha}-primase on the SV40 helicase is required for viral primosome activity, but helicase activity is dispensable. The Journal of Biological Chemistry 285:33475-33484. - 48.
Huang, S. G., K. Weisshart, I. Gilbert, and E. Fanning. 1998. Stoichiometry and mechanism of assembly of SV40 T antigen complexes with the viral origin of DNA replication and DNA polymerase alpha-primase. Biochemistry 37:15345-15352. - 49.
Hubscher, U., G. Maga, and S. Spadari. 2002. Eukaryotic DNA polymerases. Annual Review of Biochemistry 71:133-163. - 50.
Hubscher, U., H. P. Nasheuer, and J. E. Syvaoja. 2000. Eukaryotic DNA polymerases, a growing family. Trends in Biochemical Sciences 25:143-147. - 51.
Iftode, C., and J. A. Borowiec. 2000. 5' --> 3' molecular polarity of human replication protein A (hRPA) binding to pseudo-origin DNA substrates. Biochemistry 39:11970-11981. - 52.
Iftode, C., Y. Daniely, and J. A. Borowiec. 1999. Replication protein A (RPA): the eukaryotic SSB. Crit Rev Biochem Mol Biol 34:141-180. - 53.
Ishimi, Y. 1997. A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex. The Journal of Biological Chemistry 272:24508-24513. - 54.
Jiang, X., V. Klimovich, A. I. Arunkumar, E. B. Hysinger, Y. Wang, R. D. Ott, G. D. Guler, B. Weiner, W. J. Chazin, and E. Fanning. 2006. Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex. Embo J 25:5516-5526. - 55.
Kanke, M., Y. Kodama, T. S. Takahashi, T. Nakagawa, and H. Masukata. 2012. Mcm10 plays an essential role in origin DNA unwinding after loading of the CMG components. Embo J 31:2182-2194. - 56.
Katou, Y., Y. Kanoh, M. Bando, H. Noguchi, H. Tanaka, T. Ashikari, K. Sugimoto, and K. Shirahige. 2003. S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424:1078-1083. - 57.
Kelly, T. J., P. Simancek, and G. S. Brush. 1998. Identification and characterization of a single-stranded DNA-binding protein from the archaeon Methanococcus jannaschii. Proceedings of the National Academy of Sciences of the United States of America 95:14634-14639. - 58.
Kenny, M. K., S. H. Lee, and J. Hurwitz. 1989. Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: single-strand stabilization and stimulation of DNA polymerases alpha and delta. Proceedings of the National Academy of Sciences of the United States of America 86:9757-9761. - 59.
Kerr, I. D., R. I. Wadsworth, L. Cubeddu, W. Blankenfeldt, J. H. Naismith, and M. F. White. 2003. Insights into ssDNA recognition by the OB fold from a structural and thermodynamic study of Sulfolobus SSB protein. Embo J 22:2561-2570. - 60.
Khopde, S., R. Roy, and D. T. Simmons. 2008. The binding of topoisomerase I to T antigen enhances the synthesis of RNA-DNA primers during simian virus 40 DNA replication. Biochemistry 47:9653-9660. - 61.
Khopde, S., and D. T. Simmons. 2008. Simian virus 40 DNA replication is dependent on an interaction between topoisomerase I and the C-terminal end of T antigen. Journal of Virology 82:1136-1145. - 62.
Kim, C., B. F. Paulus, and M. S. Wold. 1994. Interactions of human replication protein A with oligonucleotides. Biochemistry 33:14197-14206. - 63.
Kim, S., H. G. Dallmann, C. S. McHenry, and K. J. Marians. 1996. Coupling of a replicative polymerase and helicase: a tau-DnaB interaction mediates rapid replication fork movement. Cell 84:643-650. - 64.
Kowalczykowski, S. C. 2000. Initiation of genetic recombination and recombination-dependent replication. Trends in Biochemical Sciences 25:156-165. - 65.
Kunkel, T. A., and P. M. Burgers. 2008. Dividing the workload at a eukaryotic replication fork. Trends Cell Biol 18:521-527. - 66.
Kuo, S. R., J. S. Liu, T. R. Broker, and L. T. Chow. 1994. Cell-free replication of the human papillomavirus DNA with homologous viral E1 and E2 proteins and human cell extracts. The Journal of Biological Chemistry 269:24058-24065. - 67.
Lee, C., I. Liachko, R. Bouten, Z. Kelman, and B. K. Tye. 2010. Alternative mechanisms for coordinating polymerase alpha and MCM helicase. Molecular and Cellular Biology 30:423-435. - 68.
Lee, J. K., and J. Hurwitz. 2001. Processive DNA helicase activity of the minichromosome maintenance proteins 4, 6, and 7 complex requires forked DNA structures. Proceedings of the National Academy of Sciences of the United States of America 98:54-59. - 69.
Lee, S. H., and D. K. Kim. 1995. The role of the 34-kDa subunit of human replication protein A in simian virus 40 DNA replication in vitro. The Journal of Biological Chemistry 270:12801-12807. - 70.
Lei, M., and B. K. Tye. 2001. Initiating DNA synthesis: from recruiting to activating the MCM complex. Journal of Cell Science 114:1447-1454. - 71.
Li, D., R. Zhao, W. Lilyestrom, D. Gai, R. Zhang, J. A. DeCaprio, E. Fanning, A. Jochimiak, G. Szakonyi, and X. S. Chen. 2003. Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen. Nature 423:512-518. - 72.
Lin, B. Y., A. M. Makhov, J. D. Griffith, T. R. Broker, and L. T. Chow. 2002. Chaperone proteins abrogate inhibition of the human papillomavirus (HPV) E1 replicative helicase by the HPV E2 protein. Molecular and Cellular Biology 22:6592-6604. - 73.
Liu, J. S., S. R. Kuo, A. M. Makhov, D. M. Cyr, J. D. Griffith, T. R. Broker, and L. T. Chow. 1998. Human Hsp70 and Hsp40 chaperone proteins facilitate human papillomavirus-11 E1 protein binding to the origin and stimulate cell-free DNA replication. The Journal of Biological Chemistry 273:30704-30712. - 74.
Liu, Y., H. I. Kao, and R. A. Bambara. 2004. Flap endonuclease 1: a central component of DNA metabolism. Annual Review of Biochemistry 73:589-615. - 75.
Lohman, T. M. 1992. Escherichia coli DNA helicases: mechanisms of DNA unwinding. Molecular Microbiology 6:5-14. - 76.
Loo, Y. M., and T. Melendy. 2000. The majority of human replication protein A remains complexed throughout the cell cycle. Nucleic Acids Research 28:3354-3360. - 77.
Loo, Y. M., and T. Melendy. 2004. Recruitment of replication protein A by the papillomavirus E1 protein and modulation by single-stranded DNA. Journal of Virology 78:1605-1615. - 78.
Lou, H., M. Komata, Y. Katou, Z. Guan, C. C. Reis, M. Budd, K. Shirahige, and J. L. Campbell. 2008. Mrc1 and DNA polymerase epsilon function together in linking DNA replication and the S phase checkpoint. Molecular Cell 32:106-117. - 79.
Lusky, M., J. Hurwitz, and Y. S. Seo. 1993. Cooperative assembly of the bovine papilloma virus E1 and E2 proteins on the replication origin requires an intact E2 binding site. The Journal of Biological Chemistry 268:15795-15803. - 80.
Ma, T., N. Zou, B. Y. Lin, L. T. Chow, and J. W. Harper. 1999. Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication. Proceedings of the National Academy of Sciences of the United States of America 96:382-387. - 81.
Maga, G., I. Frouin, S. Spadari, and U. Hubscher. 2001. Replication protein A as a "fidelity clamp" for DNA polymerase alpha. The Journal of Biological Chemistry 276:18235-18242. - 82.
Makiniemi, M., T. Hillukkala, J. Tuusa, K. Reini, M. Vaara, D. Huang, H. Pospiech, I. Majuri, T. Westerling, T. P. Makela, and J. E. Syvaoja. 2001. BRCT domain-containing protein TopBP1 functions in DNA replication and damage response. The Journal of Biological Chemistry 276:30399-30406. - 83.
Masterson, P. J., M. A. Stanley, A. P. Lewis, and M. A. Romanos. 1998. A C-terminal helicase domain of the human papillomavirus E1 protein binds E2 and the DNA polymerase alpha-primase p68 subunit. Journal of Virology 72:7407-7419. - 84.
Masumoto, H., A. Sugino, and H. Araki. 2000. Dpb11 controls the association between DNA polymerases alpha and epsilon and the autonomously replicating sequence region of budding yeast. Molecular and Cellular Biology 20:2809-2817. - 85.
Matson, S. W., and K. A. Kaiser-Rogers. 1990. DNA helicases. Annual Review of Biochemistry 59:289-329. - 86.
Matsumoto, T., T. Eki, and J. Hurwitz. 1990. Studies on the initiation and elongation reactions in the simian virus 40 DNA replication system. Proceedings of the National Academy of Sciences of the United States of America 87:9712-9716. - 87.
Melendy, T., J. Sedman, and A. Stenlund. 1995. Cellular factors required for papillomavirus DNA replication. Journal of Virology 69:7857-7867. - 88.
Melendy, T., and B. Stillman. 1993. An interaction between replication protein A and SV40 T antigen appears essential for primosome assembly during SV40 DNA replication. The Journal of Biological Chemistry 268:3389-3395. - 89.
Mer, G., A. Bochkarev, W. J. Chazin, and A. M. Edwards. 2000. Three-dimensional structure and function of replication protein A. Cold Spring Harbor Symposia on Quantitative Biology 65:193-200. - 90.
Mohr, I. J., R. Clark, S. Sun, E. J. Androphy, P. MacPherson, and M. R. Botchan. 1990. Targeting the E1 replication protein to the papillomavirus origin of replication by complex formation with the E2 transactivator. Science 250:1694-1699. - 91.
Moyer, S. E., P. W. Lewis, and M. R. Botchan. 2006. Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proceedings of the National Academy of Sciences of the United States of America 103:10236-10241. - 92.
Mueser, T. C., J. M. Hinerman, J. M. Devos, R. A. Boyer, and K. J. Williams. 2010. Structural analysis of bacteriophage T4 DNA replication: a review in the Virology Journal series on bacteriophage T4 and its relatives. Virol J 7:359. - 93.
Murakami, Y., and J. Hurwitz. 1993. DNA polymerase alpha stimulates the ATP-dependent binding of simian virus tumor T antigen to the SV40 origin of replication. The Journal of Biological Chemistry 268:11018-11027. - 94.
Murakami, Y., C. R. Wobbe, L. Weissbach, F. B. Dean, and J. Hurwitz. 1986. Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro. Proceedings of the National Academy of Sciences of the United States of America 83:2869-2873. - 95.
Nakaya, R., J. Takaya, T. Onuki, M. Moritani, N. Nozaki, and Y. Ishimi. 2010. Identification of proteins that may directly interact with human RPA. Journal of Biochemistry 148:539-547. - 96.
Nasheuer, H. P., D. von Winkler, C. Schneider, I. Dornreiter, I. Gilbert, and E. Fanning. 1992. Purification and functional characterization of bovine RP-A in an in vitro SV40 DNA replication system. Chromosoma 102:S52-59. - 97.
Neuwald, A. F., L. Aravind, J. L. Spouge, and E. V. Koonin. 1999. AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Research 9:27-43. - 98.
Nick McElhinny, S. A., D. A. Gordenin, C. M. Stith, P. M. Burgers, and T. A. Kunkel. 2008. Division of labor at the eukaryotic replication fork. Molecular Cell 30:137-144. - 99.
Notarnicola, S. M., K. Park, J. D. Griffith, and C. C. Richardson. 1995. A domain of the gene 4 helicase/primase of bacteriophage T7 required for the formation of an active hexamer. The Journal of Biological Chemistry 270:20215-20224. - 100.
Ott, R. D., C. Rehfuess, V. N. Podust, J. E. Clark, and E. Fanning. 2002. Role of the p68 subunit of human DNA polymerase alpha-primase in simian virus 40 DNA replication. Molecular and Cellular Biology 22:5669-5678. - 101.
Park, C. J., J. H. Lee, and B. S. Choi. 2005. Solution structure of the DNA-binding domain of RPA from Saccharomyces cerevisiae and its interaction with single-stranded DNA and SV40 T antigen. Nucleic Acids Research 33:4172-4181. - 102.
Patel, S. S., and M. M. Hingorani. 1993. Oligomeric structure of bacteriophage T7 DNA primase/helicase proteins. The Journal of Biological Chemistry 268:10668-10675. - 103.
Pavlov, Y. I., and P. V. Shcherbakova. 2010. DNA polymerases at the eukaryotic fork-20 years later. Mutation Research 685:45-53. - 104.
Piccolino, M. 2000. Biological machines: from mills to molecules. Nat Rev Mol Cell Biol 1:149-153. - 105.
Pike, J. E., R. A. Henry, P. M. Burgers, J. L. Campbell, and R. A. Bambara. 2010. An alternative pathway for Okazaki fragment processing: resolution of fold-back flaps by Pif1 helicase. The Journal of Biological Chemistry 285:41712-41723. - 106.
Pursell, Z. F., I. Isoz, E. B. Lundstrom, E. Johansson, and T. A. Kunkel. 2007. Yeast DNA polymerase epsilon participates in leading-strand DNA replication. Science 317:127-130. - 107.
Ricke, R. M., and A. K. Bielinsky. 2004. Mcm10 regulates the stability and chromatin association of DNA polymerase-alpha. Molecular Cell 16:173-185. - 108.
Rossi, M. L., V. Purohit, P. D. Brandt, and R. A. Bambara. 2006. Lagging strand replication proteins in genome stability and DNA repair. Chem Rev 106:453-473. - 109.
Sanders, C. M., O. V. Kovalevskiy, D. Sizov, A. A. Lebedev, M. N. Isupov, and A. A. Antson. 2007. Papillomavirus E1 helicase assembly maintains an asymmetric state in the absence of DNA and nucleotide cofactors. Nucleic Acids Research 35:6451-6457. - 110.
Sanders, C. M., and A. Stenlund. 1998. Recruitment and loading of the E1 initiator protein: an ATP-dependent process catalysed by a transcription factor. Embo J 17:7044-7055. - 111.
Schneider, C., K. Weisshart, L. A. Guarino, I. Dornreiter, and E. Fanning. 1994. Species-specific functional interactions of DNA polymerase alpha-primase with simian virus 40 (SV40) T antigen require SV40 origin DNA. Molecular and Cellular Biology 14:3176-3185. - 112.
Sclafani, R. A., and T. M. Holzen. 2007. Cell cycle regulation of DNA replication. Annu Rev Genet 41:237-280. - 113.
Sedman, J., and A. Stenlund. 1998. The papillomavirus E1 protein forms a DNA-dependent hexameric complex with ATPase and DNA helicase activities. J Virol 72:6893-6897. - 114.
Simmons, D. T., T. Melendy, D. Usher, and B. Stillman. 1996. Simian virus 40 large T antigen binds to topoisomerase I. Virology 222:365-374. - 115.
Speck, C., Z. Chen, H. Li, and B. Stillman. 2005. ATPase-dependent cooperative binding of ORC and Cdc6 to origin DNA. Nature Structural & Molecular Biology 12:965-971. - 116.
Stewart, L., G. C. Ireton, and J. J. Champoux. 1996. The domain organization of human topoisomerase I. The Journal of Biological Chemistry 271:7602-7608. - 117.
Stillman, B. 2005. Origin recognition and the chromosome cycle. FEBS Letters 579:877-884. - 118.
Szyjka, S. J., C. J. Viggiani, and O. M. Aparicio. 2005. Mrc1 is required for normal progression of replication forks throughout chromatin in S. cerevisiae. Molecular Cell 19:691-697. - 119.
Taljanidisz, J., R. S. Decker, Z. S. Guo, M. L. DePamphilis, and N. Sarkar. 1987. Initiation of simian virus 40 DNA replication in vitro: identification of RNA-primed nascent DNA chains. Nucleic Acids Research 15:7877-7888. - 120.
Tanaka, H., Y. Katou, M. Yagura, K. Saitoh, T. Itoh, H. Araki, M. Bando, and K. Shirahige. 2009. Ctf4 coordinates the progression of helicase and DNA polymerase alpha. Genes Cells 14:807-820. - 121.
Theobald, D. L., R. M. Mitton-Fry, and D. S. Wuttke. 2003. Nucleic acid recognition by OB-fold proteins. Annu Rev Biophys Biomol Struct 32:115-133. - 122.
Tourriere, H., G. Versini, V. Cordon-Preciado, C. Alabert, and P. Pasero. 2005. Mrc1 and Tof1 promote replication fork progression and recovery independently of Rad53. Molecular Cell 19:699-706. - 123.
Trowbridge, P. W., R. Roy, and D. T. Simmons. 1999. Human topoisomerase I promotes initiation of simian virus 40 DNA replication in vitro. Mol Cell Biol 19:1686-1694. - 124.
Tsurimoto, T., T. Melendy, and B. Stillman. 1990. Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin. Nature 346:534-539. - 125.
Tye, B. K. 1999. MCM proteins in DNA replication. Annual Review of Biochemistry 68:649-686. - 126.
Ustav, M., and A. Stenlund. 1991. Transient replication of BPV-1 requires two viral polypeptides encoded by the E1 and E2 open reading frames. Embo J 10:449-457. - 127.
Vos, S. M., E. M. Tretter, B. H. Schmidt, and J. M. Berger. 2011. All tangled up: how cells direct, manage and exploit topoisomerase function. Nat Rev Mol Cell Biol 12:827-841. - 128.
Waga, S., and B. Stillman. 1994. Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro. Nature 369:207-212. - 129.
Weisshart, K., H. Forster, E. Kremmer, B. Schlott, F. Grosse, and H. P. Nasheuer. 2000. Protein-protein interactions of the primase subunits p58 and p48 with simian virus 40 T antigen are required for efficient primer synthesis in a cell-free system. The Journal of Biological Chemistry 275:17328-17337. - 130.
Weisshart, K., P. Taneja, and E. Fanning. 1998. The replication protein A binding site in simian virus 40 (SV40) T antigen and its role in the initial steps of SV40 DNA replication. Journal of Virology 72:9771-9781. - 131.
Wilson, V. G., M. West, K. Woytek, and D. Rangasamy. 2002. Papillomavirus E1 proteins: form, function, and features. Virus Genes 24:275-290. - 132.
Wold, M. S. 1997. Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annual Review of Biochemistry 66:61-92. - 133.
Wun-Kim, K., R. Upson, W. Young, T. Melendy, B. Stillman, and D. T. Simmons. 1993. The DNA-binding domain of simian virus 40 tumor antigen has multiple functions. Journal of Virology 67:7608-7611. - 134.
Yang, L., R. Li, I. J. Mohr, R. Clark, and M. R. Botchan. 1991. Activation of BPV-1 replication in vitro by the transcription factor E2. Nature 353:628-632. - 135.
Yang, L., M. S. Wold, J. J. Li, T. J. Kelly, and L. F. Liu. 1987. Roles of DNA topoisomerases in simian virus 40 DNA replication in vitro. Proceedings of the National Academy of Sciences of the United States of America 84:950-954. - 136.
Yang, L., M. S. Wold, J. J. Li, T. J. Kelly, and L. F. Liu. 1987. Roles of DNA topoisomerases in simian virus 40 DNA replication in vitro. Proceedings of the National Academy of Sciences of the United States of America 84:950-954. - 137.
You, Z., Y. Komamura, and Y. Ishimi. 1999. Biochemical analysis of the intrinsic Mcm4-Mcm6-mcm7 DNA helicase activity. Molecular and Cellular Biology 19:8003-8015. - 138.
Yuzhakov, A., Z. Kelman, J. Hurwitz, and M. O'Donnell. 1999. Multiple competition reactions for RPA order the assembly of the DNA polymerase delta holoenzyme. Embo J 18:6189-6199. - 139.
Zhou, B., D. R. Arnett, X. Yu, A. Brewster, G. A. Sowd, C. L. Xie, S. Vila, D. Gai, E. Fanning, and X. S. Chen. 2012. Structural basis for the interaction of a hexameric replicative helicase with the regulatory subunit of human DNA polymerase alpha-primase. The Journal of Biological Chemistry. - 140.
Zhu, W., C. Ukomadu, S. Jha, T. Senga, S. K. Dhar, J. A. Wohlschlegel, L. K. Nutt, S. Kornbluth, and A. Dutta. 2007. Mcm10 and And-1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication. Genes & Development 21:2288-2299. - 141.
141.Zlotkin, T., G. Kaufmann, Y. Jiang, M. Y. Lee, L. Uitto, J. Syvaoja, I. Dornreiter, E. Fanning, and T. Nethanel. 1996. DNA polymerase epsilon may be dispensable for SV40- but not cellular-DNA replication. Embo J 15:2298-2305.