Cryo-EM Structure of the Replisome Reveals Multiple Interactions Coordinating DNA Synthesis

Cryo-EM structure of the replisome reveals multiple interactions coordinating DNA synthesis

Arkadiusz W. Kulczyka,1, Arne Moellerb,2, Peter Meyera, Piotr Sliza, and Charles C. Richardsona,1

aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; and bDepartment of Molecular Biology and Genetics, The Danish Research Institute of Translational Neuroscience, Aarhus University, 8000 Aarhus, Denmark

Contributed by Charles C. Richardson, January 27, 2017 (sent for review December 7, 2016; reviewed by James M. Berger and Nicholas E. Dixon) We present a structure of the ∼650-kDa functional replisome of with genetically altered gp4 and gp5, trx, and DNA resembling a bacteriophage T7 assembled on DNA resembling a replication fork. replication fork in buffer containing dTTP (Fig. 1A). Gp4 contains A structure of the complex consisting of six domains of DNA heli- an amino acid substitution, E343Q. Glutamate 343 functions as a case, five domains of RNA primase, two DNA polymerases, and catalytic base for hydrolysis of dTTP, an event that leads to trans- two thioredoxin (processivity factor) molecules was determined location on DNA. A crystal structure of the hexameric gp4 reveals by single-particle cryo-electron microscopy. The two molecules of that E343 is in position to activate the water molecule for a nu- DNA polymerase adopt a different spatial arrangement at the repli- cleophilic attack on the nucleoside γ-phosphate (16). Consequently, cation fork, reflecting their roles in leading- and lagging-strand syn- this altered gp4 (gp4-E343Q) binds dTTP but does not catalyze thesis. The structure, in combination with biochemical data, reveals hydrolysis on DNA to which it is tightly bound. The genetically molecular mechanisms for coordination of leading- and lagging- altered gp5 contains two amino acid substitutions, D5A and E7A, in strand synthesis. Because mechanisms of DNA replication are highly the active site of the 3′-5′ exonuclease domain. The altered protein conserved, the observations are relevant to other replication systems. exhibits a polymerization activity similar to the activity of wild-type DNA polymerase, but its exonuclease activity is reduced by a factor cryo-EM structure | replisome | coordination of leading- and of 106 (10). lagging-strands synthesis | DNA replication | DNA polymerase The DNA consists of a 77-nt oligonucleotide containing two self-complementary regions that anneal to form a tetra-loop and he reverse polarity and antiparallel nature of the two strands a 5-bp double-helical region in the middle of the sequence with a Tin duplex DNA pose a topological problem for their simul- 32-nt 5′-overhang and a 31-nt 3′-overhang (Fig. 1A). These two taneous synthesis. The “trombone” model of DNA replication overhangs mimic the lagging and the leading strand, respectively. postulates that the lagging strand forms a loop such that the Although the sequence is capable of forming intermolecular leading- and lagging-strand replication proteins contact one an- polymers as well as the desired intramolecular monomeric other (1). This replication loop contains a nascent Okazaki species, the monomeric form is favored by inclusion of the tetra- fragment and allows for coordination of leading- and lagging- loop sequence. The tetra-loop forms a highly stable turn confor- strand synthesis. The bacteriophage T7 replisome has been mation only in the monomeric form that we previously confirmed studied extensively (2). Thus, the macromolecular complex of T7 by 1D and 2D 1H-NMR (17). We assessed the folding prop- replication proteins provides an excellent model for structural erties of an oligonucleotide resembling a replication fork using analysis of a replisome. Notably, the human mitochondrial and the T7 replication systems are similar (3). Significance The phage T7 replisome is relatively simple. Four proteins are sufficient for reconstitution of the functional replisome, yet the The antiparallel nature of the two strands in duplex DNA poses assembled replisome recapitulates all of the key features of more a topological problem for their simultaneous synthesis. The complex prokaryotic and eukaryotic systems (2, 4). These pro- “trombone” model of the replication fork postulates that the teins are the following: DNA polymerase (gp5) and its proc- Escherichia coli lagging-strand forms a loop such that the leading- and lagging- essivity factor, thioredoxin (trx), single-stranded strand replication proteins contact one another. The replisome DNA (ssDNA)-binding protein (gp2.5), and the bifunctional then can move in one direction along the DNA while synthe- DNA primase-helicase (gp4). sizing both strands. Physical interactions between the replica- Gp4 forms multiple oligomeric forms (5). The negative stain tion proteins and DNA coordinate processive synthesis of the EM revealed that hexamers and heptamers are the most abun- leading and lagging strands. Here, we present the structure ∼ ∼ dant ( 22% and 78%, respectively). Upon addition of ssDNA, of a functional replisome from bacteriophage T7. Our struc- the equilibrium between the two oligomeric forms changes tural and biochemical analyses provide an explanation of the ∼ ∼ ( 77% protein rings are now hexameric, and 18% are hepta- mechanisms governing coordination of leading- and lagging- meric), indicative of DNA binding (6). The hexamer is an en- strand synthesis. zymatically active form of gp4. It binds to ssDNA, hydrolyzes nucleotides, translocates on ssDNA, and unwinds dsDNA (7–9). Author contributions: A.W.K. and C.C.R. designed research; A.W.K. performed research; In contrast, the heptamer does not bind to ssDNA (6). A.W.K., A.M., P.M., and P.S. contributed new reagents/analytic tools; A.W.K. and C.C.R. Crystal structures of all of the individual T7 replication pro- analyzed data; and A.W.K. and C.C.R. wrote the paper. teins have been determined (10–15). However, to date there is Reviewers: J.M.B., Johns Hopkins University School of Medicine; and N.E.D., University of Wollongong. no structural information on the T7 replisome or its subassemblies. Consequently, intermolecular interactions involved in coordination The authors declare no conflict of interest. of DNA synthesis have not been identified. We have therefore used Data deposition: The cryo-EM map of the replisome was deposited in the EM Data Bank, www.emdatabank.org (accession no. EMD-8565). cryo-electron microscopy (cryo-EM) and single-particle recon- 1To whom correspondence may be addressed. Email: [email protected] or ccr@hms. struction methods to determine a structure of the T7 replisome. harvard.edu. 2Present address: Department of Structural Biology, Max Planck Institute of Biophysics, Results 60538 Frankfurt, Germany. Replisome Assembly. We have identified conditions necessary to as- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. semble the T7 replication complex. A stable complex was obtained 1073/pnas.1701252114/-/DCSupplemental.

E1848–E1856 | PNAS | Published online February 21, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1701252114 Downloaded by guest on September 25, 2021 PNAS PLUS BIOPHYSICS AND COMPUTATIONAL BIOLOGY Fig. 1. cryo-EM analysis and assembly of the replisome. (A) A fork-shaped DNA is formed by folding of ssDNA such that a stable tetra-loop is formed in the middle of the sequence flanked by a short double-helical region; two oligonucleotides are annealed to serve as primers. (B and C) Melting profiles of the tetra-loop DNA. Samples containing different concentrations of the fork-shaped DNA are represented by colored curved lines. Melting curves (B) and their

first derivatives (C) show a biphasic transition with the melting temperatures (Tm)of53± 2°C.(D) Fluorescence anisotropy measurements of replisome formation. The replication complex assembled in the presence of gp4-E343Q is four times more stable than the complex assembled with wild-type gp4. Apparent binding constants are 0.2 ± 0.01 μM and 0.8 ± 0.05 μM, respectively. (E) A representative cryo-EM image. (Scale bar, 10 nm.) (F) The 2D class averages. (Scale bar, 10 nm.) (G) A calculated 3D map of the replisome. The initial model for the 3D calculation was generated using the atomic coordinates of the helicase domain of gp4 filtered to a resolution of 60 Å. (H) Fourier Schell Correlation (FSC). The resolution of 13.8 Å is based on the gold-standard FSC0.5 criterion. (I) Comparison of reprojections from the 3D structure with the 2D class averages. (Scale bar, 10 nm.)

Kulczyk et al. PNAS | Published online February 21, 2017 | E1849 Downloaded by guest on September 25, 2021 fluorescence melting spectroscopy in the presence of a fluores- The initial cryo-EM analysis revealed that ∼95% of helicase cent dye that intercalates into double-stranded DNA. The de- rings are hexameric and ∼5% are heptameric. Thus, the initial crease in fluorescence as a function of increasing temperature model for the 3D calculation was generated using the atomic suggests folding of the template and formation of the duplex coordinates of a hexameric helicase domain of gp4 [Protein (Fig. 1B). The melting profiles show a biphasic transition in- Data Bank (PDB) ID code 1CR0] filtered to a resolution of 60 Å dicative of formation of a single monomeric species in solution (Fig. 1G). Fig. 1F displays representative 2D class averages. We with a melting temperature of 53 ± 2°C(Fig.1C). In the control clearly see additional densities associated with the hexameric experiment, a similar DNA lacking a stable tetra-loop sequence ring of DNA helicase likely representing DNA polymerases does not exhibit a biphasic melting transition (Fig. 1B). The bound to the replisome. To confirm that these densities are gp5/trx 5′-overhang of the folded template contains the primase recog- complexes, we labeled thioredoxin with 5 nm Ni-NTA Nanogold nition sequence 5′-GGGTC-3′. The lagging-strand primer is a (SI Appendix, Fig. S3). Thioredoxin was altered by addition of a tetraribonucleotide complementary to the primase recognition poly-His tag at the N terminus. Gp5-trx complex was purified sequence, and it has an additional two nucleotides at the 3′-end on Ni-NTA agarose (SI Appendix, Fig. S3A) and used to prepare that base-pair with the template: a deoxyribonucleotide at position samples for EM. Negative-stain EM reveals gold particles co- 5 and a dideoxynucleotide at position 6. The dideoxynucleotide localizing with the replisome, confirming the presence of gp5/trx SI Appendix B C at the 3′-end of the primer prevents primer extension by gp5/ in the complex ( , Fig. S3 and ). We see one or two SI Appendix trx. The leading-strand primer is a 21-nt oligonucleotide, also gold particles associated with the complex ( , Fig. C containing a dideoxynucleotide at the 3′-end (Fig. 1A). S3 ). However, this labeling with gold is not quantitative, and Upon addition of gp5/trx and gp4-E343Q to the fork DNA in gold particles are observed that are not attached to the complex. the presence of dTTP, a stable replication complex is formed as We also analyzed samples of the replisome in the absence of evidenced by an increase in fluorescence anisotropy of a fluo- DNA using negative-stain EM. When DNA is not present, no rescein-labeled template. Gp4-E343Q binds to the replication binding between gp4-E343Q and gp5/trx is detected, indicating that DNA is required for assembly of the functional gp4– complex assembled on the DNA with an affinity fourfold higher – ∼ than that measured for binding of gp4 (0.2 ± 0.01 μM and 0.8 ± E343Q gp5/trx complex. In the absence of DNA, 70% helicase μ D rings are heptameric, and ∼20% are hexameric, consistent with 0.05 M, respectively), (Fig. 1 ). We assessed the binding of – individual replication proteins to the fork DNA in a filter-bind- previous negative-stain EM analysis (5 8), native gel electro- ing experiment, in which the leading-strand primer was radio- phoresis (5, 6, 21), and gel-filtration data (9). actively labeled at the 5′-end. Dissociation constants determined Structural classification with RELION resulted in two 3D for gp5/trx and gp4-E343Q are 0.07 ± 0.03 μM and 0.28 ± maps. One of these maps was then used for further refinements (Fig. 1G). The final 3D map was calculated using ∼50% (79,519) 0.05 μM, respectively (SI Appendix, Fig. S1). A stable replication particle images. The presented structure is thus representative of complex of gp4-E343Q and gp5 is formed with K of 0.2 ± d the entire dataset. The angular distribution plot is presented in 0.04 μM, consistent with fluorescence anisotropy measurements. SI Appendix, Fig. S5. A reported resolution of 13.8 Å is based We have previously characterized gp4–gp5/trx–DNA interactions on the gold-standard 0.5FSC criterion (Fig. 1H). The repro- on the lagging and leading strand using a variety of biophysical jections from the structure are in good agreement with the 2D methods (5, 18). The fork DNA was designed such that it com- class averages (Fig. 1I and SI Appendix, Fig. S6). bined DNA constructs used in these experiments. Well-defined electron densities allow for unambiguous fitting of two molecules of gp5/trx (PDB ID code 1T7P), six subunits of Preparation of the Cryo-Electron Microscopy Sample and Determination DNA helicase (PDB ID code 1CR0), and five of six subunits of the Replisome Structure. Detailed descriptions of the sample of RNA primase (PDB ID code 1NUI). Automatic docking of preparation protocols are included in Materials and Methods. atomic coordinates into the cryo-EM map was performed with First, the leading-strand primer and the template were annealed the Chimera software using the protocol described in SI Ap- by incubation at 94 °C and by slowly decreasing the temper- pendix. The cross-correlation coefficient (CCC) calculated for atureto25°C.The1.1-μM primer template was mixed with simultaneous docking of all PDBs (i.e., two copies of PDB ID 6 μM gp4-E343Q in a buffer containing 5 μM lagging-strand n β code 1T7P, five copies of PDB ID code 1NUI, and PDB ID code primer, 10 mM dTTP, and 0.001% wt/vol -dodecyl -D-maltoside. 1CR0) is 0.91. The CCCs calculated for individual protein do- Gp5/trx was added to the mixture after the 10-min incubation at mains range from 0.83 to 0.99 and are presented in SI Appendix, 25 °C. Samples of the replisome were adsorbed onto freshly glow- Fig. S7. The local CCCs mapped onto the cryo-EM structure are discharged holey carbon grids and flash-frozen in liquid ethane presented in SI Appendix, Fig. S8. using a Vitrobot with a controlled temperature and humidity. The To determine the handedness in the map of the replisome, we protein and DNA concentrations are higher than the measured D SI Appendix performed an automatic docking of atomic coordinates into a dissociation constants (Fig. 1 and ,Fig.S1), and thus mirror copy of the map across the “y–z” plane. The local CCCs favorable conditions are created on the EM grid for complex calculated for a chiral copy of the replisome map are shown in SI – formation. To confirm formation of the protein DNA complex Appendix, Fig. S8. The model presented in this article represents under these conditions, we performed a pull-down experiment an enantiomer displaying a significantly better fit than its chiral μ with a biotin-labeled fork DNA. The 1 M DNA was incubated counterpart (SI Appendix, Fig. S8). We also performed a random μ μ with 2 M gp4-E343Q and 10 M gp5/trx in the buffer described canonical tilt experiment (SI Appendix, Fig. S9). above. Replisome samples were separated on streptavidin beads and analyzed by SDS/PAGE (SI Appendix, Fig. S2). The bands Architecture of the Replisome. An overall architecture of the T7 representing gp5/trx and gp4-E343Q on the gel confirm protein replisome is presented in Fig. 2A and Movie S1. The structure binding to the biotinylated fork DNA. reveals the presence of an asymmetric hexameric ring of DNA The cryo-EM images (Fig. 1E) were collected with a Titan helicase to which two DNA polymerases bind in an asymmetric Krios cryo-EM. Subsequent structure calculation and refinement fashion. Likewise, the associated DNA primase domains of gp4 steps were performed using single- and double-precision floating- are arranged asymmetrically (Fig. 2B). Two molecules of gp5/trx point compilations of Relion-1.4. We used software and computa- are bound to the hexameric ring of gp4 at the back of the tional infrastructure maintained by SBgrid (19), as well as high- replisome relative to the direction of dsDNA unwinding by the performance computing facilities available through the Stampede helicase. Although DNA is not visible, the unique structures Supercomputer at Texas Advanced Computing Center (20). obtained suggest strongly its presence (Discussion).

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Fig. 2. Architecture of the replisome. (A) The structure reveals the presence of the hexameric ring of DNA helicase (blue), five covalently linked DNA primase domains (orange), and two gp5/trx complexes. The polymerase domain (green) and the exonuclease domain (purple) of gp5 as well as thioredoxin (yellow) are indicated. The two molecules of DNA polymerase bind to gp4 in an asymmetric fashion, reflecting their roles in leading- and lagging-strand synthesis (gp5/ trxlead and gp5/trxlag, respectively). (B) An asymmetric arrangement of RNA primases at the replisome.

Kulczyk et al. PNAS | Published online February 21, 2017 | E1851 Downloaded by guest on September 25, 2021 Asymmetric Positioning of DNA Polymerases at the Replisome. The The Interaction Between the Leading- and Lagging-Strand DNA cryo-EM structure provides evidence for the asymmetric positioning Polymerase. Gp5/trxlead and gp5/trxlag contact each other at the of the two DNA polymerases at the replisome. The two molecules replisome. The protein–protein interface is formed by a region of gp5/trx can be implicated in leading- and lagging-strand synthesis containing the α-helices G, G1, and R from the palm domain of [gp5/trxlead and gp5/trxlag, respectively (Fig. 2B)]. The interaction gp5/trxlead and by the tip of the fingers domain of gp5/trxlag (Fig. between gp5/trx and gp4 differs on the lagging and leading 3G). Taken together, the 3D map of the ∼650-kDa replisome strand. We previously reconstituted the gp4–gp5/trxlead (5) and permits an unambiguous fitting of the atomic coordinates of gp4, gp4–gp5/trxlag (18) complexes and characterized both interac- gp5/trxlead, and gp5/trxlag. tions. The biochemical analysis is consistent with the replisome Discussion structure, and thus we can determine the location of gp5/trxlead and gp5/trxlag (Fig. 2). A low-resolution structure of the gp4–gp5/ DNA in the Replisome Structure. Although we do not directly ob- trxlead complex has been determined by small-angle X-ray scat- serve the density representing DNA in the map of the replisome, tering (SAXS) (5). The position of gp4-gp5/trxlead in the SAXS the conformation of the replication proteins (gp4-E343Q, gp5/ structure is consistent with the position of one of the two gp5/trx trxlead) is compatible with the one observed in the DNA-bound molecules from the cryo-EM structure, confirming the assign- state (5–7, 9, 10). ment of gp5/trxlead. To visualize DNA in the replisome samples, we labeled a biotinylated fork DNA with quantum dots (QD) and visualized Interaction of the Leading-Strand DNA Polymerase with DNA Primase- protein–DNA complexes by negative-stain EM. A biotin is at- Helicase. Gp5/trxlead makes contacts with the two neighboring tached to the tetra-loop sequence in the DNA template (Fig. subunits of gp4 through the fingers and the exonuclease do- 1A). The DNA-QD conjugates were purified using magnetic mains. The α-helix P and the region containing a loop connecting beads coated with diethylaminoethyl cellulose (SI Appendix, Fig. the α-helix P with the β-strand 10 (residues 566–586) from the S4A). A micrograph presented in SI Appendix, Fig. S4B shows fingers domain interact with a region of DNA helicase in close QD-DNA conjugates that were negatively stained with uranyl proximity to the α-helix (residues 402–416) (Fig. 3A). Residues formate in the absence of the replication proteins. Mixing of gp4 566–586 are missing in the crystal structure of the gp5/trx primer and gp5/trx with QD-DNA results in formation of protein template (10), suggesting flexibility in this region. Interestingly, complexes that colocalize with QDs, indicative of DNA binding the loading patch of gp5 containing the basic amino acids K587, (SI Appendix, Fig. S4 C and D). This method is not quantitative, K589, R590, and R591 is located in close proximity to this region and QD-DNA is also observed distant from the replisome. (SI Appendix, Fig. S10). We previously showed that this basic DNA binding by the replisome is also supported by the fol- loading patch of gp5 interacts with the C-terminal acidic tail of lowing observations: (i) Gp4 in the structure is hexameric. Gp4 gp4 and is essential for loading gp5 at the replication fork (22). exists in multiple oligomeric forms, but only hexamers bind to The second interaction involves the primase domain from the ssDNA (5–7, 9, 21). In the absence of ssDNA, gp4 is predomi- adjacent subunit of gp4 and the region containing the α-helix E* nantly heptameric (6). (ii) No binding between gp4 and gp5/trx from the exonuclease domain of gp5/trxlead. The α-helix E* is can be detected by negative-stain EM in the absence of DNA. unique for gp5/trx in the Pol I family of DNA polymerases. (iii) We demonstrated DNA binding by the replisome using In the crystal structure of the gp5/trx primer template, trx fluorescence anisotropy measurements with a fluorescein-labeled binds to a unique 76-amino-acid loop, the thioredoxin-binding DNA (Fig. 1D), filter-binding experiments with P32-labeled DNA domain (TBD), inserted in the thumb subdomain. The cryo-EM (SI Appendix,Fig.S1), and a pull-down assay with a biotinylated structure reveals that trx contacts the TBD in an extended con- fork DNA (SI Appendix,Fig.S2). In addition, the cryo-EM map of formation. The electron density is slightly larger than a contour the replisome reveals a density located in the channel protruding level of trx (Fig. 2A), suggesting flexibility in this region, consistent through the center of the hexameric ring of DNA helicase that may with SAXS analysis of the trx–TBD interaction (23). A loop con- represent DNA (Fig. 2A). Previous EM and biochemical data in- necting α-helices H1 and H2 in the thumb domain makes contacts dicate that ssDNA binds to gp4 in this channel (5, 8, 14). The lack of with the tip of the fingers, an interaction not observed in the crystal the density representing DNA implies conformational heterogene- structure (Fig. 3B). In summary, gp5/trxlead makes numerous in- ity in the sample. For example, it is likely that ssDNA can be bound teractions with gp4. The thumb and fingers of gp5/trxlead adopt a within the central channel of gp4 by different subsets of the six conformation similar to a closed conformation of the polymerase subunits of the helicase. In support of this notion, it is difficult to active site observed upon binding of gp5/trx to a primer template in visualize the helicase–DNA complexes by cryo-EM even at high the presence of an incoming nucleotide (Fig. 3 B and C)(10). resolution (24). While our manuscript was under review, an article was pub- Interaction of the Lagging-Strand DNA Polymerase with DNA Primase- lished describing a low-resolution X-ray structure (4.8 Å) of the Helicase. Gp5/trxlag makes extensive contacts with two primase do- gp4–gp5/trx complex in the absence of DNA (25). Consistent mains from adjacent subunits in gp4 through the fingers and the with our data, gp4 in this structure is heptameric. Three mole- D exonuclease domain (Fig. 3 ). However, in contrast to gp5/trxlead, cules of gp5/trx are associated with gp4 through the C-terminal the polymearse active site of gp5/trxlag adopts an open conformation tails of DNA helicase; the interaction described earlier used (Fig. 3E). In this open conformation, the fingers rotate outward single-molecule methods (26, 27) and SAXS (5). away from the polymerase active site. The observed open conformation is consistent with the biochemical data indicating that DNA Architecture of the Replication Fork. Upon analysis of the T7 replisome primers ≥21 nt are necessary for closure of the fingers (23) and structure, in combination with the extensive biochemical and muta- supports the observation that the presence of the primase is re- tional studies reviewed in refs. 2 and 4, we propose a functional quired for stabilization of shorter primers in the polymerase active model of the replication fork (Fig. 4A). DNA helicase contacts site of gp5 (18). Interestingly, a loop connecting the α-helix P with dsDNA and separates dsDNA into ssDNA. The two ssDNAs are β α the -strand 10 and -helices E* and F from the exonuclease do- bound by gp5/trxlead and gp5/trxlag. Both DNA polymerases are po- main is also located in this interface, indicating that gp5/trxlag uses sitioned behind gp4 relative to the direction of dsDNA unwinding. similar structural motifs to contact gp4 as does gp5/trxlead.TheTBD The lagging-strand ssDNA passes through the central channel in of gp5/trxlag displays an extended conformation. As in gp5/trxlead,a the hexameric ring of DNA helicase (5, 8). The crystal structure of slightly larger electron density than a contour level of trx indicates the hexameric gp4 reveals the presence of three loops protruding flexibility in this region (Fig. 3F). into the central cavity (14). These loops contain a number of basic

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Fig. 3. Intermolecular interactions at the replisome. (A) Interaction of gp5/trxlead and gp4. (B) The fingers of gp5/trxlead interact with the loop connecting α-helices H1 and H2 in the thumb domain. (C) Gp5/trxlead adopts a closed conformation of the polymerase active site, in which the thumb and fingers are rotated inward. (D) In contrast, the polymearse active site of gp5/trxlag adopts an open conformation. In this open conformation, the fingers rotate outward away from the thumb. (E) Interaction between gp5/trxlag and gp4. (F) Trx binds to a unique 76-amino-acid loop, TBD, inserted in the thumb subdomain of gp5/trxlag. (G) Binding of gp5/trxlead and gp5/trxlag. The specific interactions are described in the text.

Kulczyk et al. PNAS | Published online February 21, 2017 | E1853 Downloaded by guest on September 25, 2021 Fig. 4. Architecture of the replication fork and a model for formation of the lagging-strand replication loop. (A) A model of the replication fork before formation of the replication loop on the lagging strand. (B) Polymerization of nucleotides by gp5/trxlag combined with the movement of ssDNA through the central cavity of gp4 accounts for formation of the replication loop on the lagging strand.

residues involved in DNA binding (16). Hydrolysis of dTTP by gp4 Appendix,Fig.S11). We showed earlier that F523 interacts di- is coupled with a sequential transfer of ssDNA by the DNA- rectly with the extruded DNA leading strand (21). The orien- binding loops from one subunit to the adjacent subunit within tation of gp5/trxlead at the replisome helps to stabilize partially the central channel (14, 16). ssDNA exiting from the channel has unwound dsDNA at the junction between ssDNA and dsDNA, the correct polarity for binding by the primase and the DNA- thus helping to separate dsDNA. Additionally, gp5/trxlead bind- binding cleft of gp5/trxlag. ing to gp4 can change the equilibrium between the forward and Displacement of the leading-strand ssDNA by the C-terminal the backward Brownian sliding of DNA helicase along ssDNA acidic surface of gp4 allows for binding of ssDNA by the positively (28, 29), favoring the movement of gp4 in the 5′-3′ direction charged active site of gp5/trxlead. In the model, a primer template or sliding of the lagging-strand ssDNA through the central boundtogp5/trxlead (PDB #1T7P) is located such that the extension channel of gp4 in the 3′-5′ direction. of the 5′-end of the template would position it in close proximity to The surface of the replisome is negatively charged with the the subunit interface β-hairpin containing residue F523 of gp4 (SI exception of regions implicated in DNA binding (SI Appendix,

E1854 | www.pnas.org/cgi/doi/10.1073/pnas.1701252114 Kulczyk et al. Downloaded by guest on September 25, 2021 Fig. S10). These regions have strong electropositive potential, ciation/association to the primer template offer a sensing mecha- PNAS PLUS namely, the central channel in the hexameric ring of gp4, gp5/trx nism, in which changes on one strand are transmitted to the other active sites, loading and exchange patches located on gp5/trx, the strand via a protein–protein interaction. For example, an inward primase domains, and trx. All these regions make contacts with rotation of the fingers in gp5/trxlag resulting from the engagement of DNA in the model. the primer by gp5/trxlag, and a subsequent extension of the primer to Our model differs from the recently proposed model for the an Okazaki fragment, would lead to disruption of the contact be- Saccharomyces cerevisiae replication fork based on a low-resolution tween gp5/trxlag and gp5/trxlead. However, upon completion of the cryo-EM map (30) of the complex between CMG helicase and the synthesis of an Okazaki fragment, gp5/trxlag disengages from the leading-strand DNA polymerase epsilon (Pol e). The model pro- primer, an event manifested by the outward rotation of the fingers. e posed by Sun et al. (30) suggests that Pol is positioned ahead of The fingers of gp5/trxlag are then in contact with gp5/trxlead,pro- the replisome, possibly functioning as the prow for unwinding of viding an interaction that could modulate leading-strand synthesis dsDNA. In contrast, in our model gp5/trxlead is positioned behind (Fig. 3G). The geometry of binding between gp5/trxlead and gp5/ the replisome relative to the direction of dsDNA unwinding. In- trxlag excludes a possibility to form a similar interaction with a third consistencies in the two models may reflect differences between copy of gp5/trx at the replication fork, explaining why only two prokaryotic and eukaryotic replication systems. DNA polymerases can be actively engaged in a coordinated DNA synthesis. Interestingly, T4 DNA polymerase forms dimers. The Formation of the Lagging-Strand Replication Loop. The loop on the fingers of T4 DNA polymerase are also involved in mediating the lagging strand implies changes in the orientation of gp5/trxlag.In protein–protein interaction (38), suggesting an analogous mecha- this new orientation, both leading- and lagging-strand synthesis nism for coordination of DNA synthesis in the phage T4 repli- occur at a single point and proceed in the same direction. How cation system. the replication loop is formed is not known. The geometry of Although absent in the replisome structure, the ssDNA-binding binding between gp5/trxlag and gp4 in the cryo-EM structure, protein (gp2.5) encoded by T7 is essential for coordination of the however, does suggest a mechanism. The interaction of gp5/trxlag synthesis of the two strands (39). In its absence the resulting with the primase domains of gp4 positions gp5/trxlag such that its Okazaki fragments are considerably shorter than those synthe- polymerization activity pushes the newly synthesized dsDNA sized in its presence (39). Gp2.5 stimulates activities of gp4 and B toward the central channel of gp4 (Fig. 4 ). Consequently, both gp5 during coordinated synthesis of DNA (39). Gp2.5 increases the polymerization activity of gp5/trxlag combined with the move- the frequency of initiation of lagging-strand synthesis greater ment of ssDNA through the central cavity of the helicase account than 10-fold (4). The acidic 26-residue C-terminal tail of gp2.5 for formation of the replication loop on the lagging strand. interacts with the basic loading patch of gp5/trxlag (26), the region where the acidic C-terminal tail of DNA helicase also binds. Coordination of Leading- and Lagging-Strand Synthesis. Coordina- A competition between these two overlapping interactions dur- tion of leading- and lagging-strand synthesis is critical for high – ing coordinated synthesis provides a switching mechanism to processivity of the replisome (5, 31 36). However, the enzymatic release gp5/trx from the replisome. Gp2.5 binding to the basic events on the lagging and leading strands are not equivalent. The lead loading patch of gp5/trxlag, located in the hinge region connect- lagging-strand DNA polymerase repeatedly dissociates from ing the palm domain and the fingers, may affect the inward and DNA and recycles to a new primer upon completion of each outward rotation of the fingers. Consequently, gp2.5 can mod- Okazaki fragment. Furthermore, one of the slowest events on the ulate binding of the fingers of gp5/trx with the palm domain of lagging strand is the recognition of the primase recognition se- lag gp5/trx , the interaction important for coordination of leading- quence, synthesis of oligoribonucleotides by the primase, and lead and lagging-strand synthesis. Because the basic loading patch of transfer to gp5/trx , which, in the time required for these events, lag gp5/trx is located next to the region involved in stabilization of would lead to accumulation of approximately 1,000 nucleotides lag gp5/trx on the replisome (SI Appendix, Fig. S10), gp2.5 binding of leading-strand DNA. Why does the synthesis of the leading lag could also regulate release of the replication loop. strand not outpace the synthesis of the lagging strand? In summary, the structure of the replisome reveals multiple Two models have been proposed to achieve coordination of interactions between DNA polymerase, DNA helicase, and RNA leading- and lagging-strand synthesis. In the first model, leading- primase. Spatial topology of binding between the individual rep- strand synthesis transiently stops allowing the completion of the lication proteins provides a structural framework that brings to- slow enzymatic events on the lagging strand. The 12-s pause al- gether multiple enzymatic activities that orchestrate coordinated lows for the relatively slow process of primer synthesis (31, 32). DNA synthesis. In the second model, coordination is achieved by different synthesis rates of the leading and the lagging strands. Contrary to Materials and Methods the results described by Lee et al. (32), the studies of the T7 A more detailed description of materials and methods used in this work can replisome by Pandey et al. (37) indicated that lagging-strand be found in SI Appendix, Supplementary Materials and Methods. DNA synthesis is 38% faster than leading-strand DNA synthesis. In addition, no pausing of replisome movement was detected Recombinant Proteins. Proteins were overproduced and purified as described during primer synthesis. previously: wild-type gp4 (40), gp4-E343Q (16), gp5/trx complex in which two The different binding modes of gp5/trxlag and gp5/trxlead to gp4 amino acids in the exonuclease domain of gp5 are replaced with alanine: are compatible with the two polymerases synthesizing DNA at D5A and E7A (10). different rates. The geometry of binding between gp5/trxlead and

gp4 indicates that the rate of leading-strand synthesis is limited cryo-EM. Samples of the replisome were adsorbed onto freshly glow-dis- BIOPHYSICS AND charged holey carbon grids (Quantifoil R1.2/1.3, Cu 400 mesh) and flash-

by the rate of dsDNA unwinding by the helicase (28). DNA COMPUTATIONAL BIOLOGY frozen in liquid ethane using a Vitrobot with a controlled temperature and synthesis by gp5/trxlag is not limited by its binding to gp4, but rather by the availability of ssDNA from the replication loop humidity. Data were acquired with a Titan Krios cryo-transmission electron mi- < − -2 (Fig. 4B). croscope (FEI) at Aarhus University, operating at 300 kV and a dose of 20 e Å using the program for the automated data collection Leginon. Images were A direct interaction between gp5/trxlag and gp5/trxlead at the collected using a GATAN 4k × 4k pixel CCD camera. A total of 3,995 images were replisome provides a mechanism for coordination of the leading- recorded at a defocus of 2–3 μm and a magnification of 50,000 times corre- and lagging-strand synthesis. Conformational changes in a structure sponding to 1.19-Å pixel size. Contrast transfer function parameters were esti- of DNA polymerase (i.e., transitions between open and closed mated with ctffind4. All subsequent data-processing steps were performed using conformations of the polymerase active site) resulting from disso- single- and double-precision floating-point compilations of Relion-1.4 (41).

Kulczyk et al. PNAS | Published online February 21, 2017 | E1855 Downloaded by guest on September 25, 2021 ACKNOWLEDGMENTS. We thank Aarhus University (Aarhus, Denmark) for This work used the facilities of the XSEDE, which is supported by National access to the EM facilities; Suavek Rucinski for assistance with Appion; and Science Foundation Grant ACI-1053575. Some of the work presented here Steven Moskowitz for assistance in preparation of figures. This work was was conducted at the National Resource for Automated Molecular Micros- supported by Grant MCB150101 from Extreme Science and Engineering copy at the Scripps Institute, San Diego, which is supported by grants from Discovery Environment (XSEDE) (to A.W.K.); Grant DFF Mobilex 4093- the National Center for Research Resources (2P41RR017573) and the Na- 00238B (to A.M.); Grant R01CA163647 from the National Cancer Institute tional Institute of General Medical Sciences (9 P41 GM103310) of the Na- (to P.S.); and Grant MCB150136 from XSEDE (to A.W.K., P.S., and C.C.R.). tional Institutes of Health.

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