Dnab Drives DNA Branch Migration and Dislodges Proteins While Encircling Two DNA Strands

Molecular Cell, Vol. 10, 647–657, September, 2002, Copyright 2002 by Cell Press DnaB Drives DNA Branch Migration and Dislodges Proteins While Encircling Two DNA Strands

Daniel L. Kaplan1,3 and Mike O’Donnell1,2 demonstrate that the protein first binds to the 5Ј single- 1Laboratory of DNA Replication stranded tail region with the DNA positioned inside the 2 Howard Hughes Medical Institute central channel. With the 3Ј tail positioned outside the Rockefeller University channel, the helicase then translocates with 5Ј to 3Ј New York, New York 10021 polarity and unwinds the double-stranded DNA. These conclusions are supported by studies of T4 gp41 and T7 gp4, leading to a model for unwinding by these helicases Summary (Figure 1A) (Ahnert and Patel, 1997; Hacker and John- son, 1997; Kaplan, 2000). DnaB is a ring-shaped, hexameric helicase that un- When T. aquaticus DnaB tracks along ssDNA and then winds the E. coli DNA replication fork while encircling encounters a duplex with no 3Ј tail, the DnaB moves one DNA strand. This report demonstrates that DnaB off the ssDNA in order to encircle both strands and can also encircle both DNA strands and then actively translocate along the dsDNA (Figure 1B) (Kaplan, 2000). translocate along the duplex. With two strands posi- Thus, when the 3Ј tail is long or bulky, the strand passes tioned inside its central channel, DnaB translocates outside of DnaB’s ring, and unwinding occurs. When with sufficient force to displace proteins tightly bound the 3Ј tail is absent, DnaB slides up over both strands to DNA with no resultant DNA unwinding. Thus, DnaB such that they pass through the central channel of the may clear proteins from chromosomal DNA. Further- protein. Because unwinding does not occur when two more, while encircling two DNA strands, DnaB can strands pass through the central channel of DnaB, it drive branch migration of a synthetic Holliday junction was unclear if this movement was by passive diffusion with heterologous duplex arms, suggesting that DnaB or active translocation. may be directly involved in DNA recombination in vivo. In this study, we find that translocation of DnaB along DnaB binds to just one DNA strand during branch mi- DNA with two strands positioned in the central channel gration. T7 phage gp4 protein also drives DNA branch is an active process, as it generates sufficient force to migration, suggesting this activity generalizes to other displace a tightly bound protein from DNA. Surprisingly, ring-shaped helicases. we find that DnaB couples this force to drive branch migration of a synthetic Holliday junction, and we have Introduction determined the mechanism of this activity. During branch migration, DnaB encircles two DNA strands, but The replicative helicase of E. coli, DnaB, is a ring-shaped binds to only one of these strands. Active translocation hexamer that encircles the lagging strand while unwind- in the 5Ј to 3Ј direction along this strand causes branch ing DNA at a replication fork (LeBowitz and McMacken, migration. 1986). Functional homologs in other systems include the T7 phage gp4 and the T4 phage gp41 proteins. Results Electron microscopy studies show that these hexameric replication fork helicases are ring-shaped with a central E. coli DnaB Does Not Unwind Duplex DNA A˚ in diameter (Dong et al., 1995; Bearing Only a 5؅ Tail 40–25ف channel of Egelman et al., 1995; Martin et al., 1998). It was previously demonstrated that T. aquaticus DnaB It has been shown by a variety of techniques that can unwind duplex DNA that resembles a replication single-stranded DNA passes through and binds to the fork, having both 5Ј and 3Ј tails on one end of the duplex central channel of these ring-shaped helicases (Egel- (forked duplex, Figure 1A) (Kaplan and Steitz, 1999). man et al., 1995; Hacker and Johnson, 1997; Kaplan, However, T. aquaticus DnaB cannot unwind duplex DNA 2000; Morris and Raney, 1999). There are two crystal that bears only a 5Ј single-stranded DNA extension, or structures of related proteins that are ring-shaped, tail (Figure 1B) (Kaplan and Steitz, 1999). To determine wherein the putative DNA binding site is inside the cen- if E. coli DnaB functions in a similar manner, duplex DNA tral channel (Singleton et al., 2000; Niedenzu et al., 2001). with a fork (Figure 1A) or with a 5Ј tail (Figure 1B) was These helicases can unwind duplex DNA in vitro if the constructed using synthetic DNA oligonucleotides. DNA resembles a replication fork. In other words, the These substrates contain a duplex region of 50 base duplex DNA must have both 5Ј and 3Ј single-stranded pairs and single-stranded tails of 30 dT. E. coli DnaB extensions, or “tails,” at one end of the duplex (Ahnert was incubated with 32P-labeled DNA substrate in the and Patel, 1997; Kaplan and Steitz, 1999; LeBowitz and presence of ATP, and the reaction was analyzed by McMacken, 1986; Richardson and Nossal, 1989). Fur- native polyacrylamide gel electrophoresis. thermore, DnaB and its homologs translocate along sin- E. coli DnaB can unwind a forked duplex (Figures 1A gle-stranded DNA and unwind duplex DNA with a dis- and 1C), but it cannot unwind a duplex that has only a tinct polarity in the 5Ј to 3Ј direction (Kaplan, 2000; 5Ј tail (Figures 1B and 1C). These results are similar to LeBowitz and McMacken, 1986; Richardson and Nossal, those observed using T. aquaticus DnaB (Kaplan and 1989). Recent studies of DnaB from Thermus aquaticus Steitz, 1999). Study of the T. aquaticus DnaB on a substrate lacking 3 Correspondence: [email protected] a3Ј tail (i.e., Figure 1B) demonstrated that the enzyme, Molecular Cell 648

passive diffusion along DNA, the movement is not coupled to ATP hydrolysis and it cannot exert force. In active translocation, the movement is coupled to ATP hydrolysis and it exerts force during movement. Next, an experiment is designed to determine if E. coli DnaB translocates on duplex DNA and to distinguish between passive and active processes.

DnaB Displaces Protein Bound to Duplex DNA with a 5؅ Tail To address whether DnaB movement along dsDNA is passive or active, we asked whether E. coli DnaB can displace a protein that binds tightly to duplex DNA. If DnaB translocation along duplex DNA is passive, it should not displace bound protein, but if DnaB movement is an active process, it may be capable of displacing protein from DNA. In the following experiment, we used EBNA1, the origin binding protein of Epstein Barr virus, as it is known to bind tightly to its canonical DNA binding site (Frappier and O’Donnell, 1991). Further- more, a modified form of EBNA1 that bears a recognition site for cAMP-dependent protein kinase, called EB- NA1PK, can be radioactively labeled while still retaining its tight DNA binding capacity (Kelman et al., 1995b). To determine if DnaB can displace protein from DNA while DnaB encircles two DNA strands, we first incubated radioactively labeled EBNA1PK protein (32P-EBNA1PK) with excess duplex DNA bearing a 5Ј tail (Figure 1D). We expect that DnaB will migrate along the ssDNA tail and then move along the duplex by encircling both DNA strands. If this translocation process is active, DnaB may displace 32P-EBNA1PK. However, the displaced 32P-EBNA1PK can simply rebind the DNA. There- fore, to detect 32P-EBNA1PK displacement, we added excess plasmid DNA that contains 24 binding sites for EBNA1 (pGEMoriP). This plasmid acts as a trap to cap- ture 32P-EBNA1PK as it is released from the 5Ј-tailed duplex DNA. The reaction was analyzed by electrophoresis in a native agarose gel, which separates substrate (32P-EBNA1PK bound to the synthetic tailed duplex) from Figure 1. DnaB Displaces Protein from DNA While Encircling One 32 PK Strand or Two Strands product ( P-EBNA1 bound to plasmid). The results 32 PK (A and B) Synthetic duplex substrates contain a 5Ј ssDNA tail that demonstrate that 59% of the P-EBNA1 is displaced serves as an assembly site for the ring-shaped DnaB hexamer. DNAs by E. coli DnaB in 16 min (Figure 1D, gel; Figure 1E, are labeled with 32P (asterisk). DnaB was incubated with substrate open circles). Furthermore, the DNA is not unwound containing: (A) fork (5Ј and 3Ј ssDNA tails) or (B) only a 5Ј ssDNA during this reaction, either in the presence or absence tail. Positions of substrate and product are indicated to the right of of 32P-EBNA1PK (data not shown). This ability to displace each native gel. tightly bound protein indicates that when DnaB translo- (C) Quantitation of results of (A) (squares) and (B) (circles). (D) DnaB displaces 32P-EBNA1PK from 5Ј-tailed duplex DNA. The cates along duplex DNA without unwinding it, the move- scheme illustrates that 32P-EBNA1PK displaced from the synthetic ment is an active process and is not simple diffusion. substrate by DnaB is trapped by a plasmid which contains 24 EBNA1 We performed several control experiments to confirm sites. that EBNA1 displacement is caused by active DnaB (E) Quantitation of the results in (D) and similar experiments. The translocation. Very little of the 32P-EBNA1PK is released oligonucleotides used to construct these substrates are detailed in from duplex DNA when ATP is not added to the reaction Supplemental Table S1 at http://www.molecule.org/cgi/content/full/ 10/3/647/DC1. (Figure 1E, open squares) or when DnaB is not added to the reaction (Figure 1E, gray diamonds). However, when the duplex DNA is forked (as in Figure 1A), DnaB upon encountering the duplex, actually transits from rapidly displaces the 32P-EBNA1PK from the DNA (Figure the ssDNA to the duplex such that both strands pass 1E, closed circles). During this reaction, the DNA is un- through the central channel (Kaplan, 2000). As T. aquat- wound, but unwinding is markedly slowed by the pres- icus DnaB moves across duplex DNA, it does not lead ence of EBNA1PK (data not shown). Protein displacement to unwound DNA products. It has not been established by DnaB during unwinding is expected because DnaB, whether the helicase, as it moves along dsDNA, does like all helicases, actively translocates along DNA during so by passive diffusion or by active translocation. In unwinding (LeBowitz and McMacken, 1986). (The rate DnaB Drives DNA Branch Migration 649

Figure 2. Encounter of DnaB with Holliday Junctions DnaB is incubated with ATP and synthetic Holliday junctions containing either (A) a 5Ј tail, (B) a fork (5Ј and 3Ј tails), (C) a 3Ј tail, or (D) no ssDNA tail. DnaB is incubated with a 5Ј-tailed Holliday junction and (E) AMP-PNP or (F) various nucleotide cofactors (8 min). The diagrams and arrows to the right and left of each gel indicate the position of each product in the gel as determined by analysis of standards in each gel.

of protein displacement is slower than that of unwinding the center are homologous; thus, spontaneous branch in the absence of protein.) When the duplex DNA bears migration may occur within this central region. However, no tails, DnaB is inefficient in displacing 32P-EBNA1PK the 19 base pairs furthest from the center of the junction (Figure 1E, closed triangles), consistent with earlier stud- are heterologous, thereby preventing spontaneous ies showing that DnaB is slow to load onto blunt-ended branch migration outside of the central region. dsDNA (Kaplan, 2000). To test DnaB for ability to drive branch migration of the Holliday junction substrate, we radiolabeled one strand to follow products in a native gel (strand 1; see DnaB Drives Branch Migration of a Synthetic Figure 2A) and then incubated the substrate with DnaB Holliday Junction and ATP. The substrate is rapidly converted to several The discovery that DnaB actively translocates along product species (Figure 2A). As will be explained further dsDNA suggested to us that DnaB might be capable of below, these products are the result of DnaB-catalyzed driving branch migration of a Holliday junction. To test branch migration. this idea, we constructed a synthetic Holliday junction We also constructed a Holliday junction with two sin- (also called four-way junction or X junction) bearing a gle-stranded tails at one end to produce a “forked” Holli- 5Ј tail composed of 30 dT (for DnaB loading) by annealing day junction (Figure 2B). The helicase activity of DnaB four oligonucleotides (Figure 2A). This synthetic Holliday should unwind strand 1 of this substrate. The results in junction (lacking the 5Ј tail) has been used to study Figure 2B show a product profile that is explained mainly proteins that catalyze branch migration of Holliday junc- by DnaB-catalyzed unwinding (see Supplemental Figure tions (Karow et al., 2000). The four duplex DNA arms S1 at http://www.molecule.org/cgi/content/full/10/3/ are each 25 base pairs, and the 6 base pairs closest to 647/DC1 for a complete discussion). Molecular Cell 650

Holliday junctions bearing only a 3Ј tail (Figure 2C) or intermediate, whereas the 1-2 hybrid is readily detected no tails at all (Figure 2D) were also tested in this assay. (data not shown). With no 5Ј single-stranded tail, DnaB cannot unwind To further support the conclusion that the reaction DNA and can only slowly load onto dsDNA. Thus, we proceeds according to the scheme in Figure 3A, Reac- expect very little activity using DnaB with these two tion II of this scheme was inhibited by reversing the substrates. Indeed, as the results of Figures 2C and polarity of the bottom, or 3-4 duplex, of this junction 2D demonstrate, very little product accumulates using (Figure 3B). To reverse the polarity of the 3-4 duplex, a these Holliday junction substrates. 5Ј-5Ј DNA connection was incorporated into strand 4 The Holliday junction used in this assay contains het- (depicted as an open circle in Figure 3B), and a 3Ј-3Ј eroduplex arms; therefore, branch migration of this sub- connection was incorporated into strand 3 (depicted as strate requires the unwinding of two duplexes (1-2 and a closed circle in Figure 3B). These modifications result 3-4) without compensatory reannealing. This activity re- in strand 4 having two 3Ј ends, and strand 3 having two quires the input of energy. To confirm that DnaB-cata- 5Ј ends. A Holliday junction with these modifications is lyzed branch migration is coupled to ATP hydrolysis, similar to that in Figure 2A, except the polarity of the we incubated the 5Ј-tailed Holliday junction with DnaB 3-4 duplex is reversed (Figure 3B). and the nonhydrolyzable analog, AMP-PNP (Figure 2E). The reversed polarity of the 3-4 duplex inhibits Reac- There was no activity, as expected. Furthermore, the tion II of Figure 3A, because now the forked region of activity profile of DnaB with various nucleotide cofactors the Reaction II substrate contains two 3Ј tails, and DnaB is similar to that previously published for unwinding (Fig- cannot unwind this substrate (Kaplan, 2000). With Reac- ure 2F), suggesting a similar energy requirement (LeBo- tion II inhibited, unannealed strand 1 cannot accumulate, witz and McMacken, 1986). There was no activity when and neither can the species composed of strands 1 and DnaB was omitted from the reaction (data not shown). 2. The gel in Figure 3B shows that these two products If DnaB can drive branch migration of a synthetic do not accumulate, supporting the reaction scheme of Holliday junction bearing a 5Ј tail, it should split the Figure 3A. substrate into two, yielding the DNA products shown in Next we designed a substrate to inhibit both Reaction Reaction I of Figure 3A. However, DnaB is also a heli- II and Reaction III, thereby leading to only the two pri- case, and these products are forked templates. There- mary products of branch migration in Reaction I of Fig- fore, if DnaB should continue, in subsequent steps, to ure 3A. We incorporated reverse polarity oligonucleo- reassociate with the products of Reaction I, it will unwind tides into the 3-4 and 2-3 duplex regions of the Holliday them (shown in Reactions II and III of Figure 3A). Further- junction, as well as GC rich DNA into the 2-3 duplex more, reannealing can occur between products that (Figure 3C). By making these modifications, the sub- contain complementary sequences. Thus, many prod- strate of Reaction III of Figure 3A has a fork with two 5Ј uct species may be created if DnaB can drive branch tails, but both strands change polarity at the duplex migration of a 5Ј-tailed Holliday junction substrate. junction. This inhibits, but does not completely block, To determine if the reaction scheme illustrated in Fig- unwinding (Kaplan, 2000). Increasing the GC content of ure 3A is the path taken by DnaB, we used several the 2-3 duplex further inhibits unwinding of this sub- approaches. First, we analyzed the time course of prod- strate. As predicted by the scheme in Figure 3A, only uct accumulation; it matches that predicted by Figure the branch migration product accumulates using this 3A. The most abundant product to appear first in the substrate (Figure 3C). time course is composed of strands 1 and 4 (Figure 2A), As further proof that the reaction follows the scheme the predicted product of branch migration (Reaction I of Figure 3A, we labeled a different strand of the 5Ј tail of Figure 3A). Later, unannealed strand 1 accumulates, junction (strand 2) and analyzed the reaction of DnaB the product of Reaction II. Still later, the species com- with this Holliday junction (Figure 3D). Note that many posed of strands 1 and 2 is visible. This species forms products are visible, but the first, most abundant prod- upon reannealing of strand 1 (the product of Reaction uct is composed of strands 2 and 3, as predicted by II in Figure 3A) with strand 2 (the product of Reaction Reaction I of Figure 3A. We then inhibited Reactions II III in Figure 3A). and III of Figure 3A as described above and found that One may argue that the branch migration product (1-4 essentially only the branch migration product (com- hybrid) is formed in two steps, direct unwinding of the posed of strands 2 and 3) is formed (Figure 3E). DNA strands, followed by reannealing. However, DnaB cannot unwind a duplex bearing only a 5Ј tail under T7 gp4B, but Not UvrD, Drives Branch Migration these conditions, as demonstrated in Figure 1. (The se- of Holliday Junctions quence of the strand bearing the 5Ј tail is identical in Next, we studied other helicases to determine if the Figures 1B and 2A). Further, we measured the reanneal- branch migration activity discovered here for DnaB gen- ing rate for strands 1 and 4 and determined the half-time eralizes to other helicases. First, we examined the T7 to be 60 min. In contrast, the half-time for reannealing gp4B helicase, a ring-shaped hexameric helicase that, of strands 1 and 2 is 1.3 min. The large difference in like DnaB, acts at a replication fork. We incubated gp4B reannealing rates is explained by the ability of strand 4 with dTTP and a 5Ј-tailed Holliday junction with reversed to form an intramolecular stem-loop structure. Further- polarity in duplexes 2-3 and 3-4 to inhibit Reactions II more, if we melt the 5Ј tail junction shown in Figure 2A and III of Figure 3A. As shown in Figure 4A, the Holliday by heating to 95ЊC for 5 min and then allow reannealing junction substrate is rapidly converted to the branch to occur at 37ЊC, the branch migration product com- migration product (1-4 hybrid). The reaction is nearly posed of strands 1 and 4 is not seen as a reannealing complete by 1 min, faster than that observed for DnaB DnaB Drives DNA Branch Migration 651

Figure 3. DnaB Catalyzes Branch Migration of a Holliday Junction (A) The scheme illustrates the proposed path of product formation. First, DnaB, while encircling two strands, catalyzes branch migration to provide duplexes 1-4 and 2-3 (Reaction I). These products contain forked ends and can be unwound by DnaB in subsequent steps, as shown in Reactions II and III. Reannealing of strands with complementary sequences can also occur (not shown). (B) DnaB action on a 5Ј-tailed Holliday junction with reversed polarity of the 3-4 duplex. The open circle represents a 5Ј-5Ј DNA connection, the closed circle represents a 3Ј-3Ј connection. (C) DnaB action on a 5Ј-tailed Holliday junction with reversed polarity of the 3-4 and 2-3 duplexes. The 2-3 duplex is GC rich. (D) DnaB action on a 5Ј-tailed Holliday junction with strand 2 labeled with 32P. (E) DnaB action on a 5Ј-tailed Holliday junction with strand 2 labeled with 32P. The 3-4 and 2-3 duplexes have reversed polarity. The 2-3 duplex is GC rich.

(Figure 3C). Thus, branch migration activity may be a We used a helicase substrate that has a 3Ј tail attached general property shared by members of this ring-shaped to the Holliday junction, since UvrD unwinding is stimu- helicase family. lated by a 3Ј tail, not a 5Ј tail (Figure 4B) (Matson, 1986). We also examined UvrD, a helicase of the SF1 super- As shown in Figure 4B, UvrD unwinds strand 4 of this family that does not form a ring-shape (Ali et al., 1999). substrate, but no branch migration product (1-4 hybrid)

Figure 4. Hexameric T7 gp4B Also Catalyzes Branch Migration, but UvrD Does Not (A) Analysis of T7 gp4B helicase on a 5Ј-tailed Holliday junction with reversed polarity of the 2-3 and 3-4 duplexes. The 2-3 duplex is GC rich. (B) Analysis of UvrD helicase with a 3Ј-tailed Holliday junction with reversed polarity of the 2-3 duplex. Molecular Cell 652

Figure 5. DnaB Encircles Both Strands 1 and 4 during Branch Migration, as Determined by Biotin/Streptavidin Blocks (A) Analysis of DnaB on a 5Ј-tailed Holliday junction (strand 2 labeled) with the biotin/ streptavidin positioned on strand 4 or strand 1. Results of product analysis on a native gel are quantified in the plot (squares, no biotin; triangles, biotin on strand 1; circles, biotin on strand 4). (B) DnaB action on a 5Ј-tailed Holliday junction, with strand 1 labeled and the biotin/ streptavidin positioned on strand 3 or strand 2 (squares, no biotin; triangles, biotin on strand 3, circles, biotin on strand 2).

is visible. Thus, branch migration activity is not a general Figure 5B). This slight decrease may be due to streptavi- property of all helicases. din blocking Reaction III of Figure 3A. With Reaction DnaB and T7 gp4B are likely acting in a processive III inhibited, reannealing of the products of Reaction I fashion during branch migration, since if these protein (Figure 3A) will occur faster, and less product will accu- rings were to dissociate from the Holliday junction dur- mulate at later time points. ing branch migration, the substrate would reanneal and We conclude that the strand bearing the 5Ј tail and no activity would be observed. Further studies to follow the strand that is complementary to it near the tail both demonstrate that the DnaB ring surrounds the duplex pass through the central channel of DnaB during branch during branch migration, consistent with a processive migration, whereas the other two strands pass outside mechanism. the DnaB ring. As a control, we performed similar experiments with the forked Holliday junction substrate, upon which DnaB During Branch Migration, Strands 1 and 4, but Not 2 mainly acts as a helicase. Unwinding is inhibited only or 3, Pass through the Central Channel of DnaB by streptavidin bound to strand 1, as expected (see We next examined which DNA strands pass through the Supplemental Figure S2 at http://www.molecule.org/ central channel of DnaB during branch migration, using cgi/content/full/10/3/647/DC1). biotin/streptavidin as a steric block. The diameter of ˚ ف streptavidin is 45 A, larger than the central channel of During Branch Migration, DnaB Binds Only DnaB helicase. Thus, streptavidin bound to a DNA to the Strand Bearing the 5؅ Tail strand should prevent it from passing through the central We next examined which strand DnaB actually binds to channel of DnaB, and movement should be blocked. In during branch migration, using reverse polarity oligonu- contrast, when streptavidin is bound to the DNA strand cleotides (i.e., having internal 3Ј-3Ј or 5Ј-5Ј linkages). If that passes on the outside of the DnaB ring, activity DnaB binds a particular strand during branch migration, should not be inhibited. it should sense the chemistry and polarity of this strand, In the experiment of Figure 5, we replaced a dT with and thus will not be able to move across a link that a biotin-dT within the test strand and then added excess reverses the strand polarity. In contrast, if DnaB does streptavidin to the reaction to create a steric block. Bio- not bind a particular strand, then its movement should tin-dT still pairs with dA on the complementary strand not be inhibited by a reversal in strand polarity. and has a spacer arm connecting biotin to dT. When In the experiments of Figure 6A, we constructed three biotin-dT is present on strand 4 (Figure 5A), branch mi- Holliday junction substrates that contain reverse polarity gration activity is abolished (open circles in the graph). linkages in either the 2-3, 3-4, or 1-2 duplex arms. Each Likewise, when biotin-dT is present on strand 1, activity of these substrates was incubated with DnaB, and the is also completely blocked (open triangles in the graph rate of product formation was compared to a reaction in Figure 5A). These data suggest that strands 1 and 4 using a substrate with no polarity reversal. of the Holliday junction pass through the central channel In our model, neither strand of the 2-3 duplex passes of DnaB. (We previously showed that DnaB can displace through the central channel of DnaB. Since the DNA a small percentage of EBNA from duplex DNA in 4 min binding site of DnaB is believed to be on the inside of [Figures 1D and 1E], but DnaB presumably does not the ring, it seems unlikely that the protein binds to either displace streptavidin here. Two likely explanations for strand 2 or strand 3. Consistent with this idea, reversing this are that streptavidin binds tighter to biotin than the polarity of the 2-3 duplex does not substantially EBNA to duplex DNA, and protein displacement during inhibit product accumulation (Figure 6A, open triangles). branch migration may be slower compared to protein (Since this polarity reversal inhibits Reaction III of Figure displacement during translocation along duplex DNA.) 3A, the reannealing rate to reform the Holliday junction In contrast, when biotin-dT is present on strand 3 will increase, and less product will accumulate at later (Figure 5B), it has no effect on branch migration (open time points.) In contrast, reversing the polarity of the 1-2 triangles in the graph). When biotin-dT is present on duplex completely abolished branch migration activity strand 2, there is a slight decrease in product accumula- (Figure 6A, open diamonds). Thus, DnaB likely makes tion at later time points (closed circles in the graph in direct contact with one of these strands. DnaB Drives DNA Branch Migration 653

Figure 6. DnaB Tracks on Only One Strand, Even Though it Encircles Two Strands DnaB action was analyzed on synthetic Holli- day junctions with different modifications. (A) Reaction rate of DnaB with 5Ј-tailed Holli- day junctions containing a 2-3 duplex reverse polarity (left diagram, triangles in graph), a 3-4 duplex reverse polarity (middle diagram, circles in graph), a 1-2 duplex reverse polarity (right diagram, diamonds in graph), or no reverse polarity (squares, no diagram shown). (B) Reaction rate of DnaB with 5Ј-tailed Holli- day junctions containing a shortened 1-2 duplex (left panel, triangles in graph) or a shortened 3-4 duplex (right panel, squares in graph). (C) Reaction rate of DnaB with 5Ј-tailed Holli- day junctions containing no hexaethylene glycol 1-phosphate (squares in graph), or hexaethylene glycol 1-phosphate within strand 2 (left panel, circles in graph) or strand 1 (right panel, triangles in graph). The zigzag line represents replacement of nine nucleotides with three hexaethylene glycol 1-phosphate groups.

Reversing the polarity of the 3-4 duplex resulted in a passes through the central channel of DnaB, it is likely modest decrease in activity (Figure 4D, closed circles). that strand 1 contacts DnaB. This idea is also consistent The 5Ј to 5Ј connection in strand 4 likely creates a steric with the ability of DnaB to translocate in the 5Ј to 3Ј block as this region of DNA passes through the central direction along the strand it loads on. channel of DnaB. This effect may be combined with an To experimentally determine which strand of the 1-2 enhanced reannealing rate (Reaction II of Figure 3A is duplex DnaB binds, nine nucleotides within either strand blocked) to give the modest inhibition observed. If DnaB were replaced with three hexaethylene glycol 1-phos- were specifically binding to either strand of the 3-4 du- phate groups. This chemical group is too small to create plex during branch migration, one may expect to see a a steric block, but its chemistry is completely different much larger decrease in activity. from DNA. Thus, if DnaB binds the strand with the hexa- To confirm that the modest decrease observed for the ethylene glycol 1-phosphate, branch migration should reverse polarity of the 3-4 duplex is not due to specific be blocked. If DnaB does not bind this strand, activity binding of this duplex by DnaB, Holliday junction sub- should not be inhibited. As expected, hexaethylene gly- strates with a short 3-4 duplex or a short 1-2 duplex col 1-phosphate within strand 1 (open triangles), but not were tested. As expected, DnaB branch migration is strand 2 (closed circles), substantially inhibited branch substantially inhibited by a shortened 1-2 duplex (open migration (Figure 6C). Thus, DnaB binds to strand 1 but triangles, Figure 6B). This result supports the conclusion not strand 2 during branch migration. that DnaB binds to the 1-2 duplex during branch migra- In conclusion, strands 1 and 4 pass through the central tion. In marked contrast, DnaB rapidly drives branch channel of DnaB during branch migration, but DnaB migration of the duplex with a short 3-4 duplex (closed binds only to strand 1. squares, Figure 6B), supporting the idea that DnaB does not bind to either strand of the 3-4 duplex. Discussion The data for reversed polarity and shortened duplexes demonstrate that DnaB binds to the 1-2 duplex, but This report reveals an activity of the ring-shaped hexa- not the 2-3 duplex or the 3-4 duplex, during branch meric helicase DnaB in which it actively translocates migration. Which strand of the 1-2 duplex does DnaB along DNA with two strands positioned within the central bind? Since strand 1, but not strand 2, of the 1-2 duplex channel. This activity allows DnaB to displace proteins Molecular Cell 654

Figure 7. DnaB Action in Branch Migration and Protein Displacement (A) Mechanism of DnaB branch migration of a 5Ј-tailed Holliday junction with heterologous duplex arms. Step i: DnaB loads onto and encircles strand 1. Step ii: DnaB slips onto the 1-4 duplex with both strands positioned in the central channel. Step iii: DnaB binds mainly to strand 1 and translocates along this strand in the 5Ј to 3Ј direction with enough force to simultaneously unwind the 1-2 and 3-4 duplexes. Step iv: once branch migration is complete, DnaB dissociates. (B) DnaB clears protein upstream from a leading strand nick (left panel) or a lagging strand lesion (right panel). Left panel: when DnaB encounters a nick in the leading strand, it may encircle both parental strands and translocate upstream. DnaB then displaces DNA- bound proteins, which may enable DNA repair proteins to initiate repair. Right panel: DnaB may bind to the lagging strand downstream from a polymerase stalled at a lesion. DnaB will translocate upstream and displace the DNA polymerase, thereby enabling proteins to repair the lesion. (C) DnaB-catalyzed branch migration near a replication fork. Left panel: in gap repair, the strand bearing the DNA lesion is paired with a sister strand via recombination (left branch of pathway) or fork regression (right branch of pathway). In the recombinative pathway, DnaB may bind to the leading strand and drive branch migration of the Holliday junction away from the replication fork, thereby allowing DNA repair. In fork regression, DnaB may bind to the lagging strand and drive DNA branch migration, thereby moving the DNA lesion away from the replication fork to allow repair. Right panel: in daughter-strand gap repair, DnaB may bind to the leading strand and drive branch migration of the Holliday junction away from the replication fork.

that are tightly bound to the DNA, without unwinding DnaB continues to track along the same strand, but now the DNA. We have also demonstrated that DnaB can both strands (strands 4 and 1) are positioned within the drive branch migration of a Holliday junction. Neither central channel of the protein (panel ii). When DnaB of these activities has been observed previously for a reaches the four-way junction, it continues to actively replication fork helicase. This report also demonstrates translocate along strand 1 in the 5Ј to 3Ј direction and that the T7 gp4B helicase can drive branch migration, essentially rips the top and bottom arms of the Holliday suggesting that the activity described for DnaB is gen- junction apart (panel iii). DnaB binds only to strand 1 eral for this family of helicases. Upon study of the mech- during this branch migration process. During this action, anism of DnaB-catalyzed branch migration, we found strand 1 is unwound from strand 2, and strand 4 is that two strands pass through the central channel of concomitantly unwound from strand 3. Furthermore, DnaB during branch migration, but DnaB binds to only strands 1 and 4 are both within the central channel of one of these strands. the protein, but they are not annealed to each other, since they are not complementary in this region. Once DnaB completes unwinding of both duplexes, the en- Model of How DnaB Drives Branch Migration zyme dissociates (panel iv). of a Holliday Junction DnaB tracks along strand 1, and thus it is easy to Combining the results herein with other studies of DnaB, imagine it being unwound from strand 2 by DnaB during we propose a model for DnaB branch migration in Figure branch migration. However, this study indicates that 7A. DnaB is known to load onto substrate DNA by encir- DnaB encircles strand 4 but does not bind it tightly for cling a 5Ј ssDNA tail (Bujalowski and Jezewwska, 2000). DNA tracking. Therefore, it is less easy to understand In Figure 7A, this 5Ј tail is strand 1 of the Holliday junction how strand 4 is unwound from strand 3, since DnaB (panel i). DnaB tracks along the ssDNA in the 5Ј to 3Ј does not contact either strand. We propose that as DnaB direction (LeBowitz and McMacken, 1986). Upon en- translocates along strand 1 and pulls it through the cen- countering the first complementary strand (strand 4), tral channel, strand 4 will be pulled indirectly by DnaB DnaB Drives DNA Branch Migration 655

due to the fact that strand 4 is paired to strand 1. This RuvAB proteins. RecG is not hexameric and does not indirect pulling of strand 4 through the central channel, form a ring (Singleton et al., 2001). RuvAB, on the other with strand 3 positioned outside the protein ring, forces hand, is hexameric, forms a ring shape, and has many the unwinding of strand 4 from strand 3. biochemical activities that are shared with DnaB (for a review, see West, 1997). As with DnaB, the RuvAB ring Unified Model of DnaB Activity can encircle two strands of DNA. Moreover, RuvAB un- It is possible that DnaB has up to four modes of active winds DNA with 5Ј to 3Ј polarity (Tsaneva et al., 1993). translocation: translocation along single-stranded DNA, One difference between RuvAB and DnaB is the way unwinding, translocation along double-stranded DNA to the proteins load onto DNA. DnaB loads onto a 5Ј single- dislodge proteins, and branch migration. However, it is stranded tail and then migrates toward the Holliday junc- likely that all of these processes occur by the same tion. RuvA binds directly to the four-way junction and mechanism. In previous work, it was described how recruits RuvB to that site (Parsons and West, 1993). A translocation along single-stranded DNA and unwinding second difference is that DnaB provides both strand occur by the same mechanism; namely, translocation separation and ATP-driven motor activities, whereas along single-stranded DNA accomplishes unwinding if RuvA has an acidic pin to achieve strand separation the second strand is positioned on the outside of the (Ariyoshi et al., 2000), while RuvB provides the ATP- protein ring, even though DnaB does not contact this motor function (Yamada et al., 2001). second strand (Kaplan, 2000). When DnaB drives branch migration of a Holliday junc- Implications for Collision of DnaB with Other DNA tion with heterologous duplex arms, it also translocates Binding Proteins In Vivo along one strand of DNA in the 5Ј to 3Ј direction (Figure In vitro work shows that the T4 gp41 protein can displace 7A). The force generated during this translocation is a biotin-streptavidin complex from single-stranded DNA capable of simultaneously unwinding two DNA du- (Morris and Raney, 1999). This suggests that when a plexes, even though the protein is binding to just one replication fork helicase translocates along single- DNA strand. It is the fact that the second strand is also strand DNA, it can exert force and dislodge proteins positioned within the central channel that results in from its path. Consistent with this idea, it was shown branch migration, even though DnaB is otherwise func- that T4 gp41, while unwinding DNA at a replication fork, tioning as an unwinding protein. Likewise, protein bound can displace RNA polymerase when the protein is tran- to DNA will not fit through the center of DnaB and may scribing on the lagging strand (Liu and Alberts, 1995), simply be driven off the duplex as DnaB translocates but not the leading strand. along it. Thus, unwinding, branch migration, and protein We have now shown that when DnaB is translocating displacement may all be catalyzed by the same mech- along DNA with two strands positioned in the central anism. channel, it can also displace proteins. In this case, the DNA is not concomitantly unwound. This task may be- Why Does DnaB Have a Ring Shape? come important if DnaB was unwinding DNA at a replica- DnaB translocates for thousands of base pairs during tion fork and encountered a nick in the leading strand, replication, and a ring shape may have evolved to in- in which case both strands of DNA would enter the crease its processivity. However, the ring shape may central channel (Figure 7B, left panel). This event could also be instrumental for DnaB to catalyze unwinding accomplish several things at once: (1) stop DNA unwind- and branch migration. A helicase separates two comple- ing, (2) allow DnaB to strip the broken DNA end of pro- mentary strands of DNA. By having one strand passing teins in preparation for repair, and (3) retain DnaB on through the central channel and one strand passing DNA as a participant in the ensuing repair process. outside the central channel, the strands are physically DnaB-catalyzed protein displacement may be impor- separated from each other. Physical separation inhibits tant in other cellular processes. For example, DnaB may reannealing of the complementary strands, thereby as- sisting the unwinding reaction. load onto the lagging strand downstream from a stalled During branch migration, two duplexes are simultane- DNA polymerase and then translocate upstream, dis- ously unwound. For either duplex, one strand is located lodging the polymerase to allow proteins to repair the within the central channel, while its complementary lesion (Figure 7B, right panel). In addition to DnaB, other strand is located outside (Figure 7A). Thus, the helicase helicases or translocases may be involved in protein ring allows for physical separation of two strands of DNA displacement in vivo. Using nucleosome templates as from their complementary partners, thereby inhibiting a model substrate for protein-bound DNA, the E. coli reannealing of both duplexes. Thus, a protein’s ring RecBCD and RuvAB proteins have been shown to dis- shape may help it accomplish branch migration. place nucleosomes as they unwind DNA (Eggleston et DNA unwinding and branch migration can be cata- al., 1995; Grigoriev and Hsieh, 1998). lyzed by proteins that do not form ring shapes. For these proteins, other mechanisms may be used to separate DnaB May Be Directly Involved in DNA the strands and ensure that once the strands are un- Recombination In Vivo wound, they do not immediately reanneal. When a replication fork encounters a DNA lesion, a recombinative repair process may be initiated to repair the Comparison to Other E. coli Proteins that Drive lesion and restart replication. The two major pathways of Branch Migration of Holliday Junctions recombinative repair include double-strand break re- There are several other E. coli proteins that drive branch pair, if the DNA lesion is a nick, and daughter-strand migration of Holliday junctions, including RecG and gap repair, if the lesion is not a nick. In both of these Molecular Cell 656

recombinative repair processes, a Holliday junction is a Gels were dried and then exposed to a Phospor-imaging screen likely DNA intermediate (for a review, see Cox, 2000). (Molecular Dynamics). For the unwinding reaction, product is de- The RuvAB and/or RecG proteins have previously been fined as single-stranded DNA. For Holliday junction reactions, products are defined by any species that migrates faster than substrate. ascribed the role of driving branch migration of these The percentage of products is calculated as follows: % products ϭ

Holliday junctions, but one can now consider that DnaB (%Ps Ϫ %P0)/(1 Ϫ %P0), where %Ps is the percent products in the

may catalyze this reaction (Figure 7C). Furthermore, sample lane, and %P0 is the percent products in the unreacted when a replication fork stalls, it may reverse or regress. substrate. This process, which may be meditated by RecG (McGlynn et al., 2001), RecA (Robu et al., 2001; Seigneur, EBNA1 Displacement Assay DNA was annealed as described above, except DNA strands were 2000), or by positive torsional strain of the genomic unlabeled and each was at 100 nM. EBNA1PK was labeled with DNA (Postow et al., 2001), creates a Holliday junction [␥-32P]ATP using cAMP-dependent protein kinase as described (Kel- structure. DnaB may drive branch migration of this Holli- man et al., 1995a). 32P-EBNA1PK was prebound to DNA substrate by day junction as well (Figure 7C). mixing 20 ␮lof32P-EBNA1PK (307 nM stock, 40,800 cpm/␮l) with Data with temperature sensitive mutations implicate 100 ␮l of DNA oligonucleotide duplex (200 nM stock), followed by Њ a role of DnaB in recombination. For example, repeated incubation at 25 C for 30 min. Reactions contained (unless otherwise stated) 40 nM DNA oligo- genes and sequences are prone to genetic rearrange- nucleotide duplex, 12 nM 32P-EBNA1PK, 160 nM pGEMoriP, 500 nM ments, including deletions. These deletions are mark- E. coli DnaB, 5 mM ATP, 5 mM creatine phosphate, 20 ␮g/ml creatine edly enhanced in dnaB mutant strains in a manner that is kinase, 20 mM Tris-HCl, 10 mM magnesium acetate, 20% glycerol, mostly recA and lexA-dependent (Saveson and Lovett, 100 ␮M EDTA, 40 ␮g/ml BSA, and 5 mM DTT (pH 8.0). Reactions 1997). Similarly, precise excision of transposon Tn10 is were incubated at 37ЊC for the indicated times, and then 6.5 ␮lof enhanced by a mutation in the dnaB gene (Nagel and 100 mM Tris-HCl, 100 mM EDTA, 15% Ficoll, and 0.25% xylene cyanol FF (pH 7.5) were added. Reactions were analyzed by a 0.7% Chan, 2000), and expansion of DNA repeats in E. coli is agarose gel using 1ϫ TBE, 100 V, and 25ЊC. The gel was dried and also stimulated by mutations in dnaB (Morag et al., then analyzed as described above. 1999). Furthermore, overproduction of DnaB in E. coli induces illegitimate recombination between short re- Acknowledgments gions of homology (Yamashita et al., 1999). Previous explanations for the phenotypes described above fo- We thank Dr. Smita Patel for supplying the T7 gp4B protein and Dr. Susan Taylor for supplying the catalytic subunit of the cAMP- cused upon the role of DnaB as a replication fork heli- dependent protein kinase. We thank Dr. Benedicte Michel and Dr. case. In light of the data presented in this report, one Justin Courcelle for critical reading of this manuscript. Thanks to may now consider that these phenotypes could be at- everyone in the O’Donnell Lab, especially Dr. Monett Librizi for purifi- tributed to a direct role of DnaB in recombination. cation of DnaB, Dr. Francisco Lopes de Saro for purification of UvrD, Dr. Dan Zhang for purification of EBNA1PK, and Dr. Irina Bruck for Experimental Procedures suggestions throughout this project. This research was supported by grant GM 62540 from the NIH and by HHMI. D.L.K. is the Leon and Proteins and DNA Toby Cooperman Fellow of the Damon Runyan Cancer Research Proteins were expressed in E. coli and purified as described: DnaB Foundation (DRG #1663). (Yuzhakov et al., 1996), EBNA1PK (Kelman et al., 1995b), and UvrD (Runyan et al., 1993). pGEMoriP plasmid contains 24 EBNA1 binding Received: May 17, 2002 sites (Frappier and O’Donnell, 1991). DNA oligonucleotides are listed Revised: July 26, 2002 in Supplemental Table S1 at http://www.molecule.org/cgi/content/ full/10/3/647/DC1. 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