FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2149090868> ?p ?o ?g. }

• W2149090868 endingPage "1127" @default.
• W2149090868 startingPage "1117" @default.
• W2149090868 abstract "The DNA tumor virus Simian virus 40 (SV40) is a model system for studying eukaryotic replication. SV40 large tumor antigen (LTag) is the initiator/helicase that is essential for genome replication. LTag recognizes and assembles at the viral replication origin. We determined the structure of two multidomain LTag subunits bound to origin DNA. The structure reveals that the origin binding domains (OBDs) and Zn and AAA+ domains are involved in origin recognition and assembly. Notably, the OBDs recognize the origin in an unexpected manner. The histidine residues of the AAA+ domains insert into a narrow minor groove region with enhanced negative electrostatic potential. Computational analysis indicates that this region is intrinsically narrow, demonstrating the role of DNA shape readout in origin recognition. Our results provide important insights into the assembly of the LTag initiator/helicase at the replication origin and suggest that histidine contacts with the minor groove serve as a mechanism of DNA shape readout. The DNA tumor virus Simian virus 40 (SV40) is a model system for studying eukaryotic replication. SV40 large tumor antigen (LTag) is the initiator/helicase that is essential for genome replication. LTag recognizes and assembles at the viral replication origin. We determined the structure of two multidomain LTag subunits bound to origin DNA. The structure reveals that the origin binding domains (OBDs) and Zn and AAA+ domains are involved in origin recognition and assembly. Notably, the OBDs recognize the origin in an unexpected manner. The histidine residues of the AAA+ domains insert into a narrow minor groove region with enhanced negative electrostatic potential. Computational analysis indicates that this region is intrinsically narrow, demonstrating the role of DNA shape readout in origin recognition. Our results provide important insights into the assembly of the LTag initiator/helicase at the replication origin and suggest that histidine contacts with the minor groove serve as a mechanism of DNA shape readout. Cocrystal structure reveals multidomain SV40 large T binding to DNA Dimeric LTag bound to origin DNA is intermediate in initiator-helicase assembly Interplay among OBD, Zn, and AAA+ domains enables recognition of origin DNA DNA shape readout can be mediated through histidine contacts with the minor groove SV40 large tumor antigen (LTag) transforms eukaryotic cells and is essential for viral DNA replication. SV40 replication involves essential cellular replication proteins, including primase and polymerase proteins (Fanning and Zhao, 2009Fanning E. Zhao K. SV40 DNA replication: from the A gene to a nanomachine.Virology. 2009; 384: 352-359Crossref PubMed Scopus (83) Google Scholar). To initiate eukaryotic DNA replication, multiple initiator proteins, such as Orc, cdc6, cdt1, and GINS, are required for origin binding and helicase recruitment/activation (Méndez and Stillman, 2003Méndez J. Stillman B. Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins.Bioessays. 2003; 25: 1158-1167Crossref PubMed Scopus (170) Google Scholar). For SV40 replication, however, LTag alone fulfills the functions of these multiple initiator proteins, i.e., origin recognition, melting, and unwinding (Simmons, 2000Simmons D.T. SV40 large T antigen functions in DNA replication and transformation.Adv. Virus Res. 2000; 55: 75-134Crossref PubMed Google Scholar). Thus, LTag is an integrated initiator and replicative helicase for DNA replication. LTag has three defined domains for replication: an origin binding domain (OBD), a Zn domain, and an AAA+ domain (Gai et al., 2004Gai D. Zhao R. Li D. Finkielstein C.V. Chen X.S. Mechanisms of conformational change for a replicative hexameric helicase of SV40 large tumor antigen.Cell. 2004; 119: 47-60Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar; Li et al., 2003Li D. Zhao R. Lilyestrom W. Gai D. Zhang R. DeCaprio J.A. Fanning E. Jochimiak A. Szakonyi G. Chen X.S. Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen.Nature. 2003; 423: 512-518Crossref PubMed Scopus (252) Google Scholar; Singleton et al., 2007Singleton M.R. Dillingham M.S. Wigley D.B. Structure and mechanism of helicases and nucleic acid translocases.Annu. Rev. Biochem. 2007; 76: 23-50Crossref PubMed Scopus (970) Google Scholar; Figure 1A). The SV40 core origin DNA for replication (ori) can be divided into two halves (Figure 1B), with each half containing two of the four 5′-GAGGC pentanucleotides (PEN1–PEN4) and an AT-rich (AT) or early palindrome (EP) region (Deb et al., 1986Deb S. DeLucia A.L. Baur C.P. Koff A. Tegtmeyer P. Domain structure of the simian virus 40 core origin of replication.Mol. Cell. Biol. 1986; 6: 1663-1670Crossref PubMed Scopus (113) Google Scholar). Each PEN can be bound by one OBD (Bochkareva et al., 2006Bochkareva E. Martynowski D. Seitova A. Bochkarev A. Structure of the origin-binding domain of simian virus 40 large T antigen bound to DNA.EMBO J. 2006; 25: 5961-5969Crossref PubMed Scopus (33) Google Scholar; Deb et al., 1987Deb S. Tsui S. Koff A. DeLucia A.L. Parsons R. Tegtmeyer P. The T-antigen-binding domain of the simian virus 40 core origin of replication.J. Virol. 1987; 61: 2143-2149Crossref PubMed Google Scholar). Each half origin supports the assembly of one LTag hexamer, and the full origin supports double hexamer formation (Mastrangelo et al., 1989Mastrangelo I.A. Hough P.V. Wall J.S. Dodson M. Dean F.B. Hurwitz J. ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication.Nature. 1989; 338: 658-662Crossref PubMed Scopus (263) Google Scholar; Valle et al., 2006Valle M. Chen X.S. Donate L.E. Fanning E. Carazo J.M. Structural basis for the cooperative assembly of large T antigen on the origin of replication.J. Mol. Biol. 2006; 357: 1295-1305Crossref PubMed Scopus (43) Google Scholar). The assembly of the LTag hexamer/double hexamer at the replication origin is coupled with ori DNA melting and unwinding (Borowiec et al., 1990Borowiec J.A. Dean F.B. Bullock P.A. Hurwitz J. Binding and unwinding—how T antigen engages the SV40 origin of DNA replication.Cell. 1990; 60: 181-184Abstract Full Text PDF PubMed Scopus (287) Google Scholar; Borowiec and Hurwitz, 1988Borowiec J.A. Hurwitz J. Localized melting and structural changes in the SV40 origin of replication induced by T-antigen.EMBO J. 1988; 7: 3149-3158Crossref PubMed Scopus (167) Google Scholar; Gai et al., 2004Gai D. Zhao R. Li D. Finkielstein C.V. Chen X.S. Mechanisms of conformational change for a replicative hexameric helicase of SV40 large tumor antigen.Cell. 2004; 119: 47-60Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar; Joo et al., 1998Joo W.S. Kim H.Y. Purviance J.D. Sreekumar K.R. Bullock P.A. Assembly of T-antigen double hexamers on the simian virus 40 core origin requires only a subset of the available binding sites.Mol. Cell. Biol. 1998; 18: 2677-2687Crossref PubMed Scopus (44) Google Scholar; Li et al., 2003Li D. Zhao R. Lilyestrom W. Gai D. Zhang R. DeCaprio J.A. Fanning E. Jochimiak A. Szakonyi G. Chen X.S. Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen.Nature. 2003; 423: 512-518Crossref PubMed Scopus (252) Google Scholar; Mastrangelo et al., 1989Mastrangelo I.A. Hough P.V. Wall J.S. Dodson M. Dean F.B. Hurwitz J. ATP-dependent assembly of double hexamers of SV40 T antigen at the viral origin of DNA replication.Nature. 1989; 338: 658-662Crossref PubMed Scopus (263) Google Scholar; Shen et al., 2005Shen J. Gai D. Patrick A. Greenleaf W.B. Chen X.S. The roles of the residues on the channel beta-hairpin and loop structures of simian virus 40 hexameric helicase.Proc. Natl. Acad. Sci. USA. 2005; 102: 11248-11253Crossref PubMed Scopus (50) Google Scholar; Sreekumar et al., 2000Sreekumar K.R. Prack A.E. Winters D.R. Barbaro B.A. Bullock P.A. The simian virus 40 core origin contains two separate sequence modules that support T-antigen double-hexamer assembly.J. Virol. 2000; 74: 8589-8600Crossref PubMed Scopus (20) Google Scholar; Valle et al., 2006Valle M. Chen X.S. Donate L.E. Fanning E. Carazo J.M. Structural basis for the cooperative assembly of large T antigen on the origin of replication.J. Mol. Biol. 2006; 357: 1295-1305Crossref PubMed Scopus (43) Google Scholar). Despite advances in characterizing the LTag helicase domain structure and the structure of individual OBDs interacting with the PEN origin sequence (Bochkareva et al., 2006Bochkareva E. Martynowski D. Seitova A. Bochkarev A. Structure of the origin-binding domain of simian virus 40 large T antigen bound to DNA.EMBO J. 2006; 25: 5961-5969Crossref PubMed Scopus (33) Google Scholar; Meinke et al., 2007Meinke G. Phelan P. Moine S. Bochkareva E. Bochkarev A. Bullock P.A. Bohm A. The crystal structure of the SV40 T-antigen origin binding domain in complex with DNA.PLoS Biol. 2007; 5: e23Crossref PubMed Scopus (50) Google Scholar), information is lacking regarding how the OBD, Zn domain, and AAA+ domain (the helicase domain) together recognize each half of the ori during the assembly of an LTag hexamer. Thus, an LTag structure containing OBD, Zn, and AAA+ domains can address the problem of the origin recognition and assembly mechanism in a way that cannot be addressed by studying the separate OBD or AAA+ helicase domains. Here we describe the crystal structure of the EP half origin bound by a dimeric LTag construct that contains OBD, Zn, and AAA+ domains. Our structure reveals several unexpected features in the protein-ori DNA interactions, including the inversion of a domain to contact ori DNA, a previously unidentified ori sequence for OBD recognition, and a particular DNA structural trait that is critical for recruiting the initiator/helicase (i.e., shape readout for DNA-protein recognition). Our results provide detailed mechanistic insights into how LTag initiator/helicase assembles around ori DNA, which should have broad implications for understanding the initiation of replication in other eukaryotic replication systems. The detailed insights into LTag-DNA binding provided by our structure reveal a critical role of histidine residues in protein-DNA recognition. We observe that the histidine residue of the AAA+ domain interacts with the ori DNA using a mechanism similar to that previously observed for arginine residues (Rohs et al., 2009Rohs R. West S.M. Sosinsky A. Liu P. Mann R.S. Honig B. The role of DNA shape in protein-DNA recognition.Nature. 2009; 461: 1248-1253Crossref PubMed Scopus (721) Google Scholar). On the basis of the analysis of all available crystal structures of protein-DNA complexes, we previously found that arginines can recognize minor groove shape through a shape-dependent electrostatic potential. Here, using cocrystal structures of other protein-DNA complexes, we demonstrate that histidines can play a similar role in DNA shape readout. The LTag construct (residues 131–627) used for cocrystallization with ori DNA contains three separable domains: the OBD, the Zn domain, and the AAA+ domain (Figure 1A, blue box; Figure S1). This LTag131-627 construct (“LTag” hereafter; Figure S2) is crystallized as a dimer in complex with the 32bp EP-half ori double-stranded (dsDNA; Figure 1B, blue box). The space group is P21, with each asymmetric unit containing two copies of the dimer-DNA complex, which allowed 2-fold noncrystallographic symmetry (NCS) averaging to yield an excellent electron density map during structural determination.Figure S2Secondary Structure of SV40 LTag131-627 and Sequence Alignment in the Region of K271 and K512/H513, Related to Figures 2 and 3Show full caption(A) Secondary structure of SV40 LTag131-627 that contains the OBD, Zn domain and AAA+ domain in the LTag-ori DNA complex. Arrows represent β strands, bars denote α helices.(B and C) Multiple sequence alignment around K271 (B) and K512/H513/N515 (C). The alignments show that K512 and H513 are absolutely conserved in polyomaviruses and the distantly related E1 helicase of papillomaviruses. K271, N515 are highly conserved only in polyomaviruses, but not in E1. Alignment was done using the alignment tool T-Coffee and viewed using Jalview.View Large Image Figure ViewerDownload (PPT) (A) Secondary structure of SV40 LTag131-627 that contains the OBD, Zn domain and AAA+ domain in the LTag-ori DNA complex. Arrows represent β strands, bars denote α helices. (B and C) Multiple sequence alignment around K271 (B) and K512/H513/N515 (C). The alignments show that K512 and H513 are absolutely conserved in polyomaviruses and the distantly related E1 helicase of papillomaviruses. K271, N515 are highly conserved only in polyomaviruses, but not in E1. Alignment was done using the alignment tool T-Coffee and viewed using Jalview. The electron density corresponding to the entire 32bp EP-ori DNA is sufficiently well featured to allow unambiguous assignment of the DNA nucleotide sequence (Figures 2A and 2B ). As a result, the orientation and register of the EP-ori DNA in the complex are well defined. The average rise and helix twist between adjacent base pairs are 3.4 Å and 35.0°, respectively, and thus are close to those reported for standard B-form DNA (3.32 Å and 35.4°; Olson et al., 1998Olson W.K. Gorin A.A. Lu X.J. Hock L.M. Zhurkin V.B. DNA sequence-dependent deformability deduced from protein-DNA crystal complexes.Proc. Natl. Acad. Sci. USA. 1998; 95: 11163-11168Crossref PubMed Scopus (882) Google Scholar). The EP-ori DNA interacts with all three LTag domains (OBD, Zn, and AAA+; Figures 2E and 2F, blue arrows), with each domain having unique binding features as described in later sections. One of the most surprising observations we made in the LTag-ori DNA complex structure was the manner in which the two OBDs of the dimer recognize the PEN sequences. Instead of the anticipated binding of PEN1 and PEN2 by OBD1 and OBD2 of the two subunits, only PEN1 is bound by OBD1, leaving PEN2 untouched (Figures 2D and 2E; OBD1 in pink, PEN1 and PEN2 labeled). Unexpectedly, OBD2 interacts with a GpC dinucleotide (G16C17/C16′G17′, termed “hidden site” hereafter; Figures 1B and 2D). Equally surprising was the finding that OBD2 is inverted by almost 180° compared with OBD1 (Figures 2D and 2F), allowing the two OBDs to bind the DNA from opposite faces with orientations inverted relative to each other (Figures 2C and S1) even though the AAA+ domains of the dimer are rotated by only ∼60° relative to each other. The flexible linker (residues 258–267) connecting the OBD and Zn domain allows this inversion of OBD2. However, structural modeling indicates that the linker is too short to allow OBD2 in the dimer to reach PEN2, even in its fully extended conformation. OBD1 interacts with PEN1 in the major groove in a fashion similar to the OBD-PEN interaction reported previously (Bochkareva et al., 2006Bochkareva E. Martynowski D. Seitova A. Bochkarev A. Structure of the origin-binding domain of simian virus 40 large T antigen bound to DNA.EMBO J. 2006; 25: 5961-5969Crossref PubMed Scopus (33) Google Scholar; Meinke et al., 2007Meinke G. Phelan P. Moine S. Bochkareva E. Bochkarev A. Bullock P.A. Bohm A. The crystal structure of the SV40 T-antigen origin binding domain in complex with DNA.PLoS Biol. 2007; 5: e23Crossref PubMed Scopus (50) Google Scholar). In particular, the loop region between α1 and β1, and residues just N-terminal of α3 together create a surface that interacts with PEN1 in the major groove (Figures 2E, 3A , and S3A). PEN1-G21A22G23G24 is in contact with R154, S152, and N153 (Figure 3A, highlighted). Additionally, the two consecutive cytosines (C24′C23′) of the reverse strand of PEN1 interact with the OBD1 protein backbone, whereas the 5′ guanine (G25′) of the complementary strand interacts with R204 (Figure 3A). These contacts, especially the bidentate hydrogen bonds between arginine and guanine bases, lead to highly specific interactions with PEN1, which is frequently used for sequence readout through base-specific contacts (Rohs et al., 2010Rohs R. Jin X. West S.M. Joshi R. Honig B. Mann R.S. Origins of specificity in protein-DNA recognition.Annu. Rev. Biochem. 2010; 79: 233-269Crossref PubMed Scopus (626) Google Scholar). Overall, 7 bp of PEN1 are recognized by OBD1 through base readout (Figure S1). The unexpected OBD2 binding to the hidden site involves substantial interactions with the major groove edges of the C16′G17′ dinucleotide and its complementary sequence, G16C17 (Figure 3B, hidden site G16C17/C16′G17′ labeled in Figures 1B and 2D). Therefore, the interactions with the hidden site are sequence specific, but with less specificity than the OBD1-PEN1 interactions. Because of the lack of a full PEN binding site for OBD2, R154 that interacts with the G21 base in OBD1 swings away from the DNA in OBD2 and forms a hydrogen bond with N227 (Figure 3B). Similarly, S152 that binds the A22 base in OBD1 swings away from the DNA in OBD2 (Figure 3B). The loop region containing A149, V150, and F151, which interact with the DNA backbone in the case of OBD1, reorients away from the DNA backbone in OBD2 (Figures 3B and S3B). Previous biochemical work showed that LTag double hexamers prefer to bind to PEN1 for the EP half ori (and PEN3 for the AT half; Joo et al., 1998Joo W.S. Kim H.Y. Purviance J.D. Sreekumar K.R. Bullock P.A. Assembly of T-antigen double hexamers on the simian virus 40 core origin requires only a subset of the available binding sites.Mol. Cell. Biol. 1998; 18: 2677-2687Crossref PubMed Scopus (44) Google Scholar). For the LTag double-hexamer assembly, PEN1 (on the EP-ori DNA) and PEN3 (on the AT-ori DNA) alone are sufficient, and PEN2 (EP-ori) and PEN3 (AT-ori) are dispensable (Joo et al., 1998Joo W.S. Kim H.Y. Purviance J.D. Sreekumar K.R. Bullock P.A. Assembly of T-antigen double hexamers on the simian virus 40 core origin requires only a subset of the available binding sites.Mol. Cell. Biol. 1998; 18: 2677-2687Crossref PubMed Scopus (44) Google Scholar). Our structural data are consistent with these results in that the LTag dimer assembly binds to PEN1 and not PEN2. It has also been shown that PEN1, PEN3, and the EP region together constitute a strong assembly unit for two head-to-head hexamers, whereas PEN2, PEN4, and the AT region constitute a weak alternative assembly unit (Sreekumar et al., 2000Sreekumar K.R. Prack A.E. Winters D.R. Barbaro B.A. Bullock P.A. The simian virus 40 core origin contains two separate sequence modules that support T-antigen double-hexamer assembly.J. Virol. 2000; 74: 8589-8600Crossref PubMed Scopus (20) Google Scholar). We were not able to obtain a crystal of dimeric LTag with the AT-half ori DNA under the tested conditions even after extensive exploration, probably reflecting a different strength of the protein interactions with the AT-half region. Interactions between the Zn domains and the EP-ori DNA occur mainly through the two DNA backbones via charge-charge interactions in the major groove (Figures 2E and 2F). The Zn domain of subunit 1 (Zn-1) contributes most of the binding interface with DNA, with a buried surface of 980 Å2 (versus 130 Å2 for subunit 2). The Zn domain of subunit 2 (Zn-2) has only one residue that binds the DNA backbone; all other interactions are through Zn-1 (residues in pink for Zn-1 and green for Zn-2 in Figure 3C). A total of 11 residues from Zn-1 contact the DNA backbones in a nonsequence-specific manner (Figure 3C). Such extensive interaction with the DNA backbone through the Zn domain may provide an anchoring point on the DNA that allows the AAA+ domain to bind the AT EP region for assembly. Unlike the interactions of the two OBDs with ori DNA at the major groove, the AAA+ domains of both subunits 1 and 2 interact with the minor groove (Figures 3D–3F). For subunit 1, the four residues at the β-hairpin tip (K512, H513, L514, and N515) and the adjacent helix (F459 on α14) contact DNA (Figure 3E). Together, these residues interact with three phosphate groups, two sugar moieties, and three bases (C8, G8′, and A7′) through a combination of hydrogen bonding and electrostatic and hydrophobic interactions. For subunit 2, the hydrophobic residues (F459 and L514) pack against the DNA backbone (Figure 3F), and charged residues K512, H513, and R456 interact with the phosphate backbones. Of particular interest is how the H513 residues of the two subunits interact with EP-ori DNA. It is evident that, for the most part, the two subunits contact the ori DNA differently (compare Figures 2E and 2F). However, the H513 residues on the β-hairpin from both subunits bind to the minor groove side by side in a nearly identical fashion with both imidazole rings lying in approximately the same plane, following the helical path of the minor groove. The N-N distance between the two H513 imidazole groups is ∼2.7 Å, a distance indicative of the formation of a hydrogen bond (Figure 4A), which may further stabilize the interactions within the ternary complex of the LTag dimer and ori DNA. Importantly, the H513 residues anchor at the position of the ori DNA that was shown to be melted upon hexamer assembly (Borowiec and Hurwitz, 1988Borowiec J.A. Hurwitz J. Localized melting and structural changes in the SV40 origin of replication induced by T-antigen.EMBO J. 1988; 7: 3149-3158Crossref PubMed Scopus (167) Google Scholar). The minor groove region bound by the two H513 side chains is narrower than its adjacent regions (Figure 4B, blue line), with a minimum width of 4.5 Å (versus 5.8 Å for the minor groove width of standard B-DNA). This narrower width of the minor groove could be induced by protein binding, or it could be an intrinsic structural feature of the ori DNA sequence. To distinguish between these two possibilities, we carried out Monte Carlo (MC) simulations (see Experimental Procedures for details) of the DNA structure using the origin sequence. The result indicates that this DNA region of the H513 contacts is characterized by an intrinsically narrow groove in the absence of protein binding (Figure 4B, green line). The negative electrostatic potential in the center of the minor groove is enhanced as the groove width decreases (Rohs et al., 2009Rohs R. West S.M. Sosinsky A. Liu P. Mann R.S. Honig B. The role of DNA shape in protein-DNA recognition.Nature. 2009; 461: 1248-1253Crossref PubMed Scopus (721) Google Scholar). The narrowed minor-groove region where the H513 residues bind has an electrostatic potential that is ∼2 kT/e more negative than the potential in the wider minor groove of adjacent regions (Figures 4A, red mesh, and 4B, red line). Thus, the binding of H513 residues to the EP-ori sequence is characterized by a shape readout mechanism whereby positively charged protein residues bind to intrinsically narrow regions of the minor groove with enhanced negative electrostatic potential (Rohs et al., 2009Rohs R. West S.M. Sosinsky A. Liu P. Mann R.S. Honig B. The role of DNA shape in protein-DNA recognition.Nature. 2009; 461: 1248-1253Crossref PubMed Scopus (721) Google Scholar, Rohs et al., 2010Rohs R. Jin X. West S.M. Joshi R. Honig B. Mann R.S. Origins of specificity in protein-DNA recognition.Annu. Rev. Biochem. 2010; 79: 233-269Crossref PubMed Scopus (626) Google Scholar). After observing the origin recognition mode of the LTag His513 residue in this structure, we sought to determine whether the observation that histidine residues recognize narrow minor groove widths and enhanced negative electrostatic potential is of a more general nature. We analyzed the minor groove width and electrostatic potential for various structures that are part of the IFN-β enhanceosome (Escalante et al., 2007Escalante C.R. Nistal-Villán E. Shen L. García-Sastre A. Aggarwal A.K. Structure of IRF-3 bound to the PRDIII-I regulatory element of the human interferon-beta enhancer.Mol. Cell. 2007; 26: 703-716Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar; Panne et al., 2004Panne D. Maniatis T. Harrison S.C. Crystal structure of ATF-2/c-Jun and IRF-3 bound to the interferon-beta enhancer.EMBO J. 2004; 23: 4384-4393Crossref PubMed Scopus (134) Google Scholar, Panne et al., 2007Panne D. Maniatis T. Harrison S.C. An atomic model of the interferon-beta enhanceosome.Cell. 2007; 129: 1111-1123Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar). This analysis revealed that conserved histidine residues from IRF-3 (His40) and IRF-7 (His46) that intrude into the minor groove consistently bind regions of narrow minor groove and enhanced negative electrostatic potential (Figure S4). Our LTag dimer-DNA cocrystal structure reveals how the multidomain of LTag that contains OBD, Zn, and AAA+ domains in a single polypeptide is arranged when it binds to DNA. The structure reveals that, besides the orientation difference between the two OBDs, the two subunits also show different relative orientations between the Zn and AAA+ domains (Figures 5A–5D). In particular, α10 of subunit 1 (magenta in Figure 5A), a long helix connecting the Zn and AAA+ domain, shifts away from the Zn domain by 6 Å compared with the conformations in nucleotide-free, ADP-bound, and ATP-bound states (Figure 5A), whereas α10 of subunit 2 is rotated toward the Zn domain by 13° compared with other LTag states (Gai et al., 2004Gai D. Zhao R. Li D. Finkielstein C.V. Chen X.S. Mechanisms of conformational change for a replicative hexameric helicase of SV40 large tumor antigen.Cell. 2004; 119: 47-60Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). These displacements of α10 alter the interactions between the Zn and AAA+ domains (Figures 5B and 5C), resulting in different degrees of rotation of the AAA+ domain, which brings the β-hairpins of both subunits into close proximity to insert into the minor groove of the EP-region of DNA (Figures 5A and 5H, boxed region). Also, the positions of the β-hairpins move upward toward the Zn domain even more so when compared with those in various nucleotide states (Figure 5A, boxed), which suggests that ori DNA binding induces conformational changes even greater than those caused by nucleotide binding in the LTag hexamer. Interestingly, some residues buried within the interface between neighboring subunits as shown in previous LTag hexamer structures are now exposed on the dimer’s outer surface and form contacts with DNA. For example, N515 and K271 of subunit 2 are buried at the dimer interface with subunit 1, and interact with subunit 1 in the hexamer. However, N515 and K271 of subunit 1 are on the exposed dimer surface and interact with DNA (Figures 3C and 3D). Conversely, R456 of subunit 1 is part of the dimer interface that interacts with subunit 2 (Figure 5H), but R456 of subunit 2 is located on the exposed surface and utilized for DNA binding (Figure 3F). Thus, these protein residues that are involved in protein-protein interactions for hexamer formation can also be used for DNA binding during helicase assembly. Such dual binding to either another protein subunit or the DNA of these LTag residues may have implications for the assembly of LTag subunits on ori DNA. In the LTag dimer-ori DNA complex, the DNA is in B-form conformation. The obviously narrower width of the minor groove where the β-hairpins bind appears to be a pre-existing structural feature of the ori DNA (Figure 4). No obvious deformations of the DNA are induced by LTag binding. This observation is similar to previous findings from the crystal structures of archaeal ORC initiator-dsDNA complexes (Dueber et al., 2007Dueber E.L. Corn J.E. Bell S.D. Berger J.M. Replication origin recognition and deformation by a heterodimeric archaeal Orc1 complex.Science. 2007; 317: 1210-1213Crossref PubMed Scopus (118) Google Scholar; Gaudier et al., 2007Gaudier M. Schuwirth B.S. Westcott S.L. Wigley D.B. Structural basis of DNA replication origin recognition by an ORC protein.Science. 2007; 317: 1213-1216Crossref PubMed Scopus (115) Google Scholar), which revealed no severe deformation or melting of DNA. In contrast, biochemical studies of E1 helicase/initiator, a distant homolog of LTag from papillomavirus, suggested that a trimer assembly intermediate is capable of melting ori DNA (Schuck and Stenlund, 2005Schuck S. Stenlund A. Assembly of a double hexameric helicase.Mol. Cell. 2005; 20: 377-389Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). No trimer assembly for LTag has ever been observed. However, given the LTag dimer assembly intermediate observed here, we asked whether the stable dimer intermediate could be captured in vitro and, if so, whether such a dimeric intermediate would be similar to E1 in terms of its ability to melt ori DNA. To address this question, we designed specific mutations to capture a stable dimer intermediate of LTag in solution, as it was previously shown that wild-type (WT) LTag only exists in either a stable monomeric or hexameric form, and no stable dimer or other intermediate oligomers can be observed in solution (Gai et al., 2004Gai D. Zhao R. Li D. Finkielstein C.V. Chen X.S. Mechanisms of conformational change for a replicative hexameric helicase of SV40 large tumor antigen.Cell. 2004; 119: 47-60Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar; Li et al., 2003Li D. Zhao R. Lilyestrom W. Gai D. Zhang R. DeCaprio J.A. Fanning E. Jochimiak A. Szakonyi G. Chen X.S. Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen.Nature. 2003; 423: 512-518Crossref PubMed Scopus (252) Google Scholar). Two mutants (mut1: L286D/R567E, and mut2: V350E/P417D) are designed to introduce mutations on only one surface of each mutant within the interface, so that intersubunit interactions are disrupted (Figure 6A). We predict" @default.
• W2149090868 created "2016-06-24" @default.
• W2149090868 creator A5009539262 @default.
• W2149090868 creator A5010411185 @default.
• W2149090868 creator A5019996279 @default.
• W2149090868 creator A5033987725 @default.
• W2149090868 creator A5037720590 @default.
• W2149090868 creator A5073683938 @default.
• W2149090868 date "2013-04-01" @default.
• W2149090868 modified "2023-10-18" @default.
• W2149090868 title "Mechanism of Origin DNA Recognition and Assembly of an Initiator-Helicase Complex by SV40 Large Tumor Antigen" @default.
• W2149090868 cites W1510004437 @default.
• W2149090868 cites W1552242374 @default.
• W2149090868 cites W1605567462 @default.
• W2149090868 cites W1802695076 @default.
• W2149090868 cites W1933777541 @default.
• W2149090868 cites W1980145808 @default.
• W2149090868 cites W1989075626 @default.
• W2149090868 cites W1989396934 @default.
• W2149090868 cites W1992848029 @default.
• W2149090868 cites W2007071268 @default.
• W2149090868 cites W2012627860 @default.
• W2149090868 cites W2014717496 @default.
• W2149090868 cites W2021534482 @default.
• W2149090868 cites W2031000298 @default.
• W2149090868 cites W2036681286 @default.
• W2149090868 cites W2038174431 @default.
• W2149090868 cites W2041758713 @default.
• W2149090868 cites W2046817214 @default.
• W2149090868 cites W2049221234 @default.
• W2149090868 cites W2050269324 @default.
• W2149090868 cites W2051054937 @default.
• W2149090868 cites W2052450400 @default.
• W2149090868 cites W2058390082 @default.
• W2149090868 cites W2059531322 @default.
• W2149090868 cites W2095994064 @default.
• W2149090868 cites W2098188064 @default.
• W2149090868 cites W2105439835 @default.
• W2149090868 cites W2110562802 @default.
• W2149090868 cites W2133250480 @default.
• W2149090868 cites W2143048739 @default.
• W2149090868 cites W2150350500 @default.
• W2149090868 cites W2151485475 @default.
• W2149090868 cites W2154363390 @default.
• W2149090868 cites W2155844132 @default.
• W2149090868 cites W2164021415 @default.
• W2149090868 cites W2166755632 @default.
• W2149090868 cites W2167096616 @default.
• W2149090868 doi "https://doi.org/10.1016/j.celrep.2013.03.002" @default.
• W2149090868 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3748285" @default.
• W2149090868 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/23545501" @default.
• W2149090868 hasPublicationYear "2013" @default.
• W2149090868 type Work @default.
• W2149090868 sameAs 2149090868 @default.
• W2149090868 citedByCount "46" @default.
• W2149090868 countsByYear W21490908682013 @default.
• W2149090868 countsByYear W21490908682014 @default.
• W2149090868 countsByYear W21490908682015 @default.
• W2149090868 countsByYear W21490908682016 @default.
• W2149090868 countsByYear W21490908682017 @default.
• W2149090868 countsByYear W21490908682018 @default.
• W2149090868 countsByYear W21490908682019 @default.
• W2149090868 countsByYear W21490908682020 @default.
• W2149090868 countsByYear W21490908682021 @default.
• W2149090868 countsByYear W21490908682022 @default.
• W2149090868 countsByYear W21490908682023 @default.
• W2149090868 crossrefType "journal-article" @default.
• W2149090868 hasAuthorship W2149090868A5009539262 @default.
• W2149090868 hasAuthorship W2149090868A5010411185 @default.
• W2149090868 hasAuthorship W2149090868A5019996279 @default.
• W2149090868 hasAuthorship W2149090868A5033987725 @default.
• W2149090868 hasAuthorship W2149090868A5037720590 @default.
• W2149090868 hasAuthorship W2149090868A5073683938 @default.
• W2149090868 hasBestOaLocation W21490908681 @default.
• W2149090868 hasConcept C104317684 @default.
• W2149090868 hasConcept C121332964 @default.
• W2149090868 hasConcept C161223559 @default.
• W2149090868 hasConcept C185592680 @default.
• W2149090868 hasConcept C552990157 @default.
• W2149090868 hasConcept C55493867 @default.
• W2149090868 hasConcept C62520636 @default.
• W2149090868 hasConcept C67705224 @default.
• W2149090868 hasConcept C70721500 @default.
• W2149090868 hasConcept C86803240 @default.
• W2149090868 hasConcept C89611455 @default.
• W2149090868 hasConcept C95444343 @default.
• W2149090868 hasConceptScore W2149090868C104317684 @default.
• W2149090868 hasConceptScore W2149090868C121332964 @default.
• W2149090868 hasConceptScore W2149090868C161223559 @default.
• W2149090868 hasConceptScore W2149090868C185592680 @default.
• W2149090868 hasConceptScore W2149090868C552990157 @default.
• W2149090868 hasConceptScore W2149090868C55493867 @default.
• W2149090868 hasConceptScore W2149090868C62520636 @default.
• W2149090868 hasConceptScore W2149090868C67705224 @default.
• W2149090868 hasConceptScore W2149090868C70721500 @default.
• W2149090868 hasConceptScore W2149090868C86803240 @default.
• W2149090868 hasConceptScore W2149090868C89611455 @default.
• W2149090868 hasConceptScore W2149090868C95444343 @default.

• next