Regulation of DNA Replication by Hexameric Helicases

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PERSPECTIVE

Two heads are better than one: regulation of DNA replication by hexameric helicases

Robert. A. Sclafani,1 Ryan J. Fletcher,1,2 and Xiaojiang S. Chen1,2,3

1Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA; 2Molecular and Computational Biology, University of Southern California, Los Angeles, California 90089, USA

DNA replication is tightly regulated in a cell cycle to antigen (LTag) or papillomavirus early gene product 1 ensure the integrity of genomic information during suc- (E1), which are mostly monomeric or dimeric in the in- cessive passages. The replication process can be divided active state (Dean et al. 1992; Sedman et al. 1997; Sand- into three major steps as follows: the initial assembly of ers and Stenlund 2000; Gai et al. 2004a). However, to prereplication complex (pre-RC) at the replication origin, become active for DNA replication, these helicase pro- the distortion of the origin (or origin melting) for repli- teins need to be assembled and loaded onto origin DNA cation initiation, and the elongation phase during DNA as ring-shaped oligomers that encircle the dsDNA (Wes- synthesis. In this process, long stretches of double- sel et al. 1992; Fouts et al. 1999; Valle et al. 2000). stranded DNA (dsDNA) must be unzipped in a relatively Another example is the eukaryotic minichromosome short time window within a cell growth cycle. The maintenance (MCM) protein complex that contains six daunting task of unzipping is carried out by a class of paralogs numbered MCM2–MCM7 (Tye 1999; Lei and efficient molecular machines called helicases, which are Tye 2001). The six MCM proteins can be in a trimer, shown to be ring-shaped oligomers. Here, we will focus dimer, and monomer conformation when they are not on the current understanding of the replicative helicases functioning as the helicase for DNA replication (Ishimi involved in cellular and viral DNA replication in eukary- 1997; Schwacha and Bell 2001; Davey et al. 2003). Early otic cells. work in Schizosaccharomyces pombe fission yeast showed that purified MCM2–MCM7 complexes formed a ring-shaped structure consistent with a hexamer unit General features of helicases for DNA replication of stoichiometry (meaning a 1:1 ratio of six proteins to Sophisticated mechanisms have evolved to achieve the form a hexamer; Adachi et al. 1997; Lee and Hurwitz tight regulation of DNA replication. One important level 2000). Many Archaeal species represent simpler replica- of regulation occurs during the ordered assembly of pro- tive helicase systems, as they have only one MCM gene teins to the origin to form the pre-RC complex. A key that is homologous to the eukaryotic genes. However, step in this process is the recruitment and assembly of the single MCM protein can form homo-oligomers (Kel- the ring-shaped replicative helicase at the origin of rep- man et al. 1999), and evidence suggests that hexamers lication, the active form of which can melt the origin and and double hexamers are likely to be the active helicase initiate the formation of DNA replication forks. The rep- form (Chong et al. 2000; Fletcher et al. 2003). Interest- licative helicases in eukaryotic systems, regardless of ingly, EM studies of the Methanobacterium thermoau- cellular or viral origin, all contain an AAA+ module in totrophicum MCM (mtMCM) showed a haptameric ring the helicase domain for ATP binding and hydrolysis structure (Yu et al. 2002), albeit hexameric structure is (Neuwald et al. 1999), which is essential for converting observed (Pape et al. 2003). The haptameric form is also the chemical energy of ATP to the mechanical forces for observed for T7 helicase (Toth et al. 2003). The presence unwinding duplex DNA. of the seven-subunit ring at least suggests some plastic- The helicase proteins from various replication systems ity in assembly, although its physiological relevance have different structural and biochemical states, most of awaits further studies. which are not active as the helicase for replication. One example is the relatively well-characterized viral repli- Replicative helicase: cative helicases in eukaryotic cells, such as SV40 large T a double-headed molecular machine

3Corresponding author. Double-hexamer architecture emerges as the common E-MAIL [email protected] or [email protected]; FAX feature for several replicative helicases in eukaryotic sys- (303) 315-8113. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ tems. Such double hexamers are shown to be in a head– gad.1240604. head configuration (Weisshart et al. 1999; Valle et al.

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Sclafani et al.

2000; Gomez-Lorenzo et al. 2003), that is, the two terminal OBD that recognizes and binds replication ori- hexamers contact each other through the N-terminal do- gin, and a C-terminal helicase domain that has an activ- mains in the middle of the double hexamer, with the ity of nonsequence-specific DNA binding. Not surpris- helicase ring locating on the two ends and pointing away ingly, like SV40 LTag, papillomavirus helicase protein from each other (Fletcher et al. 2003; Gomez-Lorenzo et E1 was observed to form the bilobed structure as re- al. 2003). If the helicase rings on both ends of the double vealed by EM studies, suggestive of a double-hexamer hexamer are active in unwinding DNA simultaneously, architecture as well (Fouts et al. 1999; Lin et al. 2002). the helicase is a bona-fide double-headed molecular ma- Similar to LTag, the assembly of the double hexamer at chine. the origin is regulated by ATP (Sanders and Stenlund The double hexamer as the functional form was first 1998). However, one unique feature for E1 helicase as- suggested for LTag through biochemical, genetics and sembly is the participation of another factor, a viral pro- EM studies (Mastrangelo et al. 1989; Simmons 2000). tein called E2 (discussed in detail below). LTag is in a monomeric state that equilibrates with a In prokaryotic cells, no direct physical evidence exists hexameric form in solution (Dean et al. 1992; Li et al. for a double-hexameric helicase in DNA replication. The 2003; Gai et al. 2004a). Monomeric LTag, which con- DnaB helicase has been observed to be hexameric on the tains an origin-binding domain (OBD) that recognizes basis of studies using biochemical and EM approaches. the replication origin of SV40, can bind the origin se- However, because replication in prokaryotes is also bi- quence and assemble into a double hexamer in the pres- directional, and there is no strong evidence to the con- ence of ATP. This assembly does not involve any acces- trary, a double-hexamer architecture for DnaB helicase sory proteins factors (such as a loader). The double- during replication could not be ruled out at this time hexamer assembly triggers origin distortion/melting and (Kornberg and Baker 1992). becomes active in unwinding the duplex DNA when ATP and Mg++ are available (Borowiec et al. 1991). Ob- Regulation of helicase activity though assembly viously, the conversion of the inactive monomeric LTag and activation into the active double-hexameric helicase at the origin provides an opportunity for regulating the helicase activ- The helicase for DNA replication is regulated through ity spatially and structurally. It is worth noting that the recruitment of the proteins to the origin for assem- LTag single hexamers also posses helicase activity bling into structures that are poised for unwinding ac- (Smelkova and Borowiec 1997; Alexandrov et al. 2002; tion. Nature has invented a variety of mechanisms to Uhlmann-Schiffler et al. 2002; Gai et al. 2004a). How- regulate the recruitment, assembly, and activation of he- ever, double hexamers have about 15 times higher heli- licases for replication. In eukaryotic cells, this process case activity than single hexamers. More importantly, starts from the origin recognition and binding by the LTag mutants defective in hexamer–hexamer interac- ORC complex, followed by Cdt1 and CDC6, which sub- tions fail to replicate DNA (Weisshart et al. 1999; Bar- sequently recruit the replicative helicase MCM and load baro et al. 2000), indicating the requirement for the it onto the origin DNA (Bailis and Forsburg 2004; Pras- double-hexamer architecture, albeit the basis for such a anth et al. 2004; Fig. 1A). The recruited MCM complex at requirement is unclear yet. the origin is initially inactive and becomes activated The crystal structure of the N-terminal half of mtMCM through the actions of CDK and DDK kinases. For SV40 confirms the double-hexamer architecture for the first virus that replicates in eukaryotic host cells, LTag uti- time at the molecular level. It also extends the case for lizes its OBD to specifically target the helicase to the the double-hexameric architecture to eukaryotic cells viral replication origin for the assembly of an active (Fletcher et al. 2003). The mtMCM structure reveals the double-hexameric helicase (Fig. 1B; Arthur et al. 1988; detailed molecular interactions for the double-hexamer Borowiec et al. 1990). Thus, LTag bypasses the cellular formation, which is also in a head–head configuration, regulatory factors such as Cdt1/CDC6 and kinases for like that seen for LTag. Using the mtMCM structure as recruitment, loading, and activation, allowing it to self- a guide, multiple-sequence alignment showed convinc- assemble into an active helicase, a mechanism necessary ingly that the structures of the poorly conserved N-ter- for the efficient replication of viral DNA. minal half of MCM proteins from various organisms are Yet, a different mechanism for regulating the assembly conserved, strongly suggesting that MCM in different of a replicative helicase at the origin is described by Ab- eukaryotic cells may also function as a double hexamer bate et al. (2004) in the previous issue of Genes & De- for DNA replication. velopment. The authors report the X-ray structure of a Another relatively well-characterized helicase for complex containing the papillomavirus E1 and E2 pro- DNA replication in eukaryotes is the papillomavirus E1, teins (Fig. 1C). The crystal structure of E1–E2 complex about which extensive biochemical and structural data contains the ATPase domain of E1 helicase and the ac- have been obtained. Papillomavirus and polyomavirus tivation domain of E2. E2 protein is a transcription factor (including SV40) are closely related, and the similarity that contains an activation domain at the N terminus between them ranges from the virus particle structures and a DNA-binding/dimerization domain at the C ter- (both with T = 7 icosahedron symmetry) to the genomic minus. The DNA-binding domains of both E1 and E2 organization. Even the sequence of the helicases from recognize particular sequences at the replication origin, the two viruses are homologous, both containing an N- and the binding sites for E1 and E2 are juxtaposed to

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Regulation of replicative helicases

allow the simultaneous binding of the two full-length E1 and E2 complex with much increased specificity for the origin (Mohr et al. 1990; Sedman and Stenlund 1995; Chen and Stenlund 2000). In the full-length version of E1

and E2, two copies of E1–E2 (E12–E22) may form a stable heterotetramer for targeting the origin in a highly specific manner (Chen and Stenlund 1998). In the crystal structure of the complex, E1–E2 forms a 1:1 heterodimer complex, which reflects the E1–E2 interactions in the

full-length E12–E22 heteroteramer complex. The structure of the E1–E2 complex of Abbate et al. (2004) reveals three important features that provide sig- nificant insights into how papillomavirus regulates its helicase assembly at the replication origin. The first feature is that the ATPase domain of E1 can be modeled into a hexamer that is very similar to that of LTag helicase domain. The similarity of the structures between E1 and LTag permits the modeling of the E1 monomer into the LTag hexamer structure by superpositioning the E1 ATPase domain with that of the LTag. The modeled E1 hexamer also shows the very similar ␤-hairpin structure seen in LTag hexameric central channel (Gai et al. 2004b). Evidence suggests that these ␤-hairpins may be important for DNA-binding and helicase activity, possibly for translocating and unwinding the dsDNA (Abbate et al. 2004; Gai et al. 2004b; Reese et al. 2004). A similar ␤-hairpin structure is also present in the central channel of mtMCM N-terminal double hexamer (Fletcher et al. 2003), despite the gross difference in the overall folding of MCM and the viral helicases. The second feature revolves around the E1–E2 interaction. According to the E1 hexamer model, E2 binds to E1 near the hexamerization interface, and thus should prevent E1 hexamer formation (Fig. 1D). On the basis of the E1–E2 structure, as well as on previous structural Figure 1. The different modes of replicative helicase assembly and biochemical data, Abbatte et al. (2004) proposed a at the origin of replication in three different systems. In each molecular mechanism of how E2 regulates the assembly panel, the yellow oval represents monomeric helicase proteins of E1 into the hexameric helicase. At first look, this regu- that can hexamerize and double hexamerize. (A) The recruit- lation appears to be purely inhibitory. E2 prevents E1 ment and assembly of MCM for eukaryotic DNA replication. from forming a hexamer by binding to the oligomeriza- The MCM is loaded onto the origin marked by ORC complex. tion interface. However, through its specific DNA-bind- MCM may assemble into a double-hexamer configuration at the ing activity at the origin, E2 enhances E1’s specific DNA origin. The eventual activation of MCM is regulated by CDK binding to the origin (Sedman et al. 1997; Stenlund and DDK in a cell-cycle-dependent manner. (B) The assembly of 2003), thus playing a shuttling role, or matchmaker role, SV40 LTag monomer on the origin sequence to form a double for targeting E1 to the origin of replication for latter hexamer, an active form of the helicase that melts the origin and unwinds two replication forks bidirectionally. This process events of the conversion to an active helicase, a process is ATP-dependent. (C) The assembly of papillomavirus E1 at the still not well understood. Considering the role of E2 in viral origin. The initial origin binding is accomplished by an helicase assembly at the origin, E2 can also be regarded

E12–E22 complex. The subsequent assembly to a double- as an E1 helicase loader, the equivalent of which does hexamer form depends on the successive addition of E1 to the not exist for SV40 LTag. initial complex and requires ATP binding. (D) Regulation of E1 This regulation of E1 helicase by the association with helicase assembly by E2 and ATP (structure picture kindly pro- E2 may also be important for preventing the intrinsic vided by Abbatte et al. 2004). The structure of two neighboring nonsequence-specific DNA binding by E1 helicase at E1 molecules (colored in blue) are positioned as they would be nonorigin locations (Stenlund 2003; Abbate et al. 2004). in a modeled hexamer. E2 (in green, red, and yellow) is bound to The matchmaker role may be particularly critical in one of the E1 molecules and sterically clashes with the adjacent E1 molecule. The unique loop 2 is labeled, and the location helping E1 to find the origin of viral DNA, because both where ATP would bind is shown. In this configuration, the base E1 and viral DNA are at very low abundance at the be- of ATP would clash with loop 2, and consequently, ATP binding ginning of the viral infection. The E1–E2 complex binds is expected to lead to a reorganization of loop 2 and disruption to the origin with greatly increased affinity and specific- of the E1:E2 complex, allowing the E1 hexamer to form. ity, possibly achieved through cooperative binding of E1

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Sclafani et al. and E2 at the origin (Mohr et al. 1990; Sanders and Sten- at the origin, a process that requires ATP binding, but lund 1998). Once bound to the origin, E2 finishes its not hydrolysis. The data were obtained by using perman- matchmaker task, and continued association with E1 ganate as a probe for unpaired nucleotides (Borowiec et will inhibit the assembly of E1 into an active helicase. al. 1991). This method has later been applied to detect The results of Abbate et al. (2004) reveal a third impor- the origin melting in other replicative helicase systems, tant feature that shows the interplay between E2–E1 such as papillomavirus E1 (Sanders and Stenlund 1998) binding/dissociation and E1 assembly. The dissociation and yeast MCM (Geraghty et al. 2000). of E2 from the origin has been postulated to be an ATP- The crystal structures of LTag helicase domain have dependent process (Sanders and Stenlund 1998), but the provided structural insights into a possible molecular E1–E2 complex structure by Abatte et al. (2004) has pro- mechanism of how LTag helicase can untwist/distort vided the structural basis for a molecular mechanism for and melt the origin through an iris-like motion (Li et al. an ATP-dependent E2 dissociation and E1 assembly. 2003). The structures obtained from different crystalli- Here, ATP binding serves as an allosteric effector to zation conditions, as well as in different nucleotide-bind- change the conformation of E1 and disengage E2 (Fig. 1D; ing states (Gai et al. 2004b), reveal an iris-like motion of see also Figs. 6, 7 in Abbate et al. 2004), permitting the the hexameric rings of LTag helicase. In this iris-like ATP-dependent hexamerization/double hexamerization motion, the two tiers of the hexameric helicase untwist/ of E1 into the active helicase form for replication. twist relative to each other slightly (permitted by the gap Regulation of replication through the recruitment of a between the two tiers and the long-spring like ␣-helix helicase to the origin is actually first described in the connecting the two tiers), and the central hexameric classical case of a prokaryotic virus system, the coli- channel expands/constricts like an iris of the eye re- phage ␭. Coliphage ␭ does not have its own replicative sponding to the change of light. This iris-like motion helicase, and sequesters the host hexameric helicase likely generates distortion and melting of the duplex DnaB for its genomic replication (Mallory et al. 1990). dsDNA bound to the central channel. Similar to the papillomavirus system, a virus protein ␭P The predicted regions to be distorted and melted in the binds the DnaB to form a complex and then targets it to iris model are the regions bound by the helicase domain the viral origin of replication. However, the helicase ac- of the double hexamer. These regions correspond to the tivity of DnaB recruited is inhibited in the ␭P–DnaB-␭O AT-rich and EP region of SV40 origin, which agrees well complex at the origin, even though the helicase is al- with the earlier biochemical results (Borowiec et al. ready in a hexameric form (Alfano and McMacken 1991). In this mechanism, the central OBD domain is 1989a). In this case, however, the helicase is activated by proposed to function only for origin binding, and not for removing the inhibitory ␭P from the complex, an action origin melting, a conclusion supported by prior studies carried out by host heat-shock proteins, leading to the (Chen et al. 1997; Li et al. 2003). In this respect, the initiation of DNA replication (Alfano and McMacken functions of the helicase domain for origin melting and 1989b). The ␭ phage wins the competition for recruiting replication fork unwinding are separated from that of the DnaB to the viral origin (instead of the host origin) OBD that specializes in targeting LTag to the origin. through the higher affinity of ␭P to DnaB, as well as the In the case of papillomavirus E1, unlike LTag, origin high affinity of the ␭P–DnaB complex to the viral origin. untwisting and melting may be directly linked to the In this regard, the recruitment of MCM helicase to the OBD of E1 (Enemark et al. 2000, 2002). Binding of a origin by Cdc6 and Cdt1 in the formation of eukaryotic single OBD dimer of E1 to the origin is reported to cause pre-RC complex also is similar (Stillman 1994). How- local distortion at the origin (Sanders and Stenlund ever, in eukaryotes, the activation of the MCM helicase 1998). This distortion becomes even more pronounced is modulated through another level of regulation by the when the dimer is converted to tetramers, and the fur- actions of CDK and DDK protein kinases, which may be ther assembly of E1-OBD into the hexamer/double important for linking the DNA replication to the cell hexamer likely causes the ultimate melting at the origin. cycles through cyclin-dependent kinases (Waga and Still- For eukaryotic cells, it is not yet clear what role the man 1998; Forsburg 2004). MCM helicase plays in the melting of the origin. The ORC complex, the equivalent of DnaA from Escherichia coli, is also thought to play a role in origin melting. In E. The helicase action after assembly and activation coli, DnaA protein distorts and melts the origin DNA Following the assembly and activation of the helicase, is (oriC) to reveal small ssDNA regions before the DnaB the opening and unwinding of the duplex DNA to ini- helicase unwinds the DNA (Kornberg and Baker 1992). tiate DNA replication bidirectionally. Compared with However, recent evidence in eukaryotic system suggests what is learned about the recruitment and assembly of that, like the case in SV40 LTag, MCM may perform the the helicases at the origin, little is known about how actual melting of the origin. In yeast, permanganate sen- origin DNA is melted and how double strands are un- sitivity at the ARS1 origin is only detected after DDK wound. In this respect, SV40 LTag is the best-studied action in S phase (Geraghty et al. 2000). The mcm5-bob1 system so far, due to the contributions from many labo- mutation, which presumably produces a constitutively ratories over the past decades. Early studies have shown active MCM helicase without DDK action (Sclafani et al. convincingly what regions of the origin DNA were un- 2002), produces the permanganate sensitivity even in G1 twisted/distorted and melted after the assembly of LTag phase, when the DNA is not being replicated. The phos-

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Regulation of replicative helicases phorylation may allow the MCM2–MCM7 complex to undergo a conformational change that can distort and melt the origin DNA. It is also not clear as to how the MCM helicase per- forms origin melting and fork unwinding upon activation. Nonetheless, two different mechanisms are proposed. The first is that the multiple MCM complexes located at a distance from both sides of the origin act as rotary motors to melt the origin and unwind the replication forks (Laskey and Madine 2003). The second is that MCM proteins are located at the origin and function in a similar way as proposed for LTag (Li et al. 2003). Additional mechanisms likely exist. Regardless, the recent evidence of MCM possessing origin-melting function provides a unifying theme for the dual roles of origin Figure 2. Presence of side channels on the wall of SV40 LTag melting and replication fork unwinding by all eukaryotic and mtMCM hexamers. (A) The surface representation of LTag replicative helicases. helicase domain at 2.7 Å resolution, showing the interior chamber of the helicase and the hole on the side wall (side channel; Li et al. 2003). The large chamber in the center (∼68 Å wide) is Double hexamer vs. bidirectional replication wide enough for strand separation. Blue areas are positively fork unwinding charged surfaces, red represents negatively charged surface. (B) During DNA replication, the propagation of the bidirec- A cut-away view of the surface representation of the electron density for the X-ray structure of LTag helicase domain filtered tional replication forks is traditionally described as two to 20 Å resolution, showing the same side channel as in A.(C) DNA forks departing away from each other, with one A cut-away view of the surface representation of the EM recon- active helicase complex on each growing fork. However, struction of a mtMCM hexamer (Pape et al. 2003), showing the it appears that this traditional picture of two departing similar side channel as in LTag. (D) A cartoon representation of forks needs to be reconsidered as the evidence for a a looping model, showing the pulling of dsDNA into the double- double hexamer as the active helicase emerges. In addi- hexamer helicase, the separation of dsDNA inside the chamber tion to LTag, the double-hexamer structure of mtMCM, of the helicase domain, and the exit of the separated ssDNA as together with other structural and biochemical informa- loops from the side channel (Li et al. 2003). (E) An alternative tion, raises the possibility of a double-hexamer architec- topology for the looping model in D, showing the loops on both ture for the replicative helicase for cellular DNA repli- sides of the double hexamer exit from two side channels at the same time (Gai et al. 2004b). cation in Archaeal and Eukaryotic cells. This possibility also made imminent a challenge to resolve the paradox as to how a double-hexamer helicase unwinds two rep- Concluding remarks lication forks bidirectionally. The recent crystal structures of Ltag, as well as mtMCM, As the understanding of DNA replication progresses, in- in combination with other biochemical and structural creasing evidence points to a common picture of a evidence (Borowiec et al. 1990; Wessel et al. 1992; double-hexamer architecture for replication helicases in Smelkova and Borowiec 1998), prompted us to propose a eukaryotic cells and viral systems. This is quite inter- looping-model topology to resolve the paradox of a esting, given the fact that the viral and cellular proteins double-hexamer helicase and two propogating replica- have low-sequence homology and show obvious differ- tion forks (Li et al. 2003). Two key observations in the ences in the detailed structural folds (Enemark et al. LTag structure provide support for this looping model. 2000; Fletcher et al. 2003; Li et al. 2003; Abbate et al. One is the presence of a positively charged side-channel 2004). The double-hexamer architecture may be the con- on the wall of LTag hexamer (Fig. 2A,B), and the second sequence of constraint for a common function required is the large, highly positively charged chamber seen in- for a replicative helicase, that is, origin melting and bi- side the helicase. The side-channels are present in directional fork unwinding. The different sequences and mtMCM (Fig. 2C; Fletcher et al. 2003; Pape et al. 2003) structural folds may reflect the difference in the regula- and likely to be present in the hexamers of papillomavi- tion of helicases and interactions with other replication rus E1 and other replicative helicases. In the proposed proteins in their respective systems. looping model, ssDNA loops out from the side channel The currently available information obtained by the in two possible topologies (Fig. 2D,E; Li et al. 2003; Gai combination of X-ray crystallography, NMR, and bio- et al. 2004b), whereas the dsDNA is pulled into the he- chemical/genetic approaches has greatly facilitated our licase domain of the double hexamer by the action of the understanding of the helicase functions during DNA rep- ATP-binding and hydrolysis (Gai et al. 2004b). In the lication. The high-resolution structures determined for LTag helicase domain, the interior chamber is suffi- different helicases from various systems also provide a ciently wide for separating dsDNA into ssDNA and can basis for more detailed functional studies through site- provide exit for the unwound ssDNA through the side directed mutagenesis. So far, the high-resolution struc- channel located the widest point of the interior chamber. tures of helicase domain and OBD have been determined

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Sclafani et al. for LTag, mtMCM, and papilloma virus E1 as separate antigen on Thr 124 selectively promotes double-hexamer entities. In the future, one challenge is to understand formation on subfragments of the viral core origin. J. Virol. how the different parts and domains are organized in an 74: 8601–8613. intact molecule, which should allow a better under- Borowiec, J.A., Dean, F.B., Bullock, P.A., and Hurwitz, J. 1990. standing of the helicase function for origin melting and Binding and unwinding—How T antigen engages the SV40 origin of DNA replication. Cell 60: 181–184. replication fork unwinding in different systems. Because Borowiec, J.A., Dean, F.B., and Hurwitz, J. 1991. Differential the replicative helicases for DNA replication in higher induction of structural changes in the simian virus 40 origin + eukaryotes belong to the AAA protein family that con- of replication by T antigen. J. Virol. 65: 1228–1235. stitutes a group of hexameric molecular machines func- Chen, G. and Stenlund, A. 1998. Characterization of the DNA- tioning in various cellular processes (helicase, protein binding domain of the bovine papillomavirus replication ini- unfolding, membrane fusion, etc.), the studies of these tiator E1. J. Virol. 72: 2567–2576. helicases should also contribute to the understanding of ———. 2000. Two patches of amino acids on the E2 DNA bind- the mechanisms of other AAA+ hexameric molecular ing domain define the surface for interaction with E1. J. Vi- machines. rol. 74: 1506–1512. Although our focus is on the discussion of helicase Chen, L., Joo, W.S., Bullock, P.A., and Simmons, D.T. 1997. The N-terminal side of the origin-binding domain of simian virus function and its regulation, we should bear in mind that 40 large T antigen is involved in A/T untwisting. J. 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Two heads are better than one: regulation of DNA replication by hexameric helicases

Robert. A. Sclafani, Ryan J. Fletcher and Xiaojiang S. Chen

Genes Dev. 2004, 18: Access the most recent version at doi:10.1101/gad.1240604

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