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Genes to Cells (2005) 10, 13-22. doi:10.1111/j.1365-2443.2004.00812.x
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Human Werner helicase interacting protein 1 (WRNIP1) functions as a novel modulator for DNA polymerase {delta}

Toshiki Tsurimoto1,*, Ayako Shinozaki2,a, Masaki Yano2, Masayuki Seki3 and Takemi Enomoto3

1 Department of Biology, School of Sciences, Kyushu University, Fukuoka 812-8581, Japan
2 Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan
3 Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Human WRNIP1, a Werner DNA helicase interacting protein 1, was expressed in insect cells and E. coli. The purified protein behaved as a homo-oligomeric complex with a native molecular mass indicative of an octamer, and the complex copurified with an ATPase activity that was stimulated by double-stranded DNA ends. As suggested by genetic studies of budding yeast WRNIP1/Mgs1, the purified human WRNIP1 complex interacted physically with human DNA polymerase {delta} (pol {delta}), stimulating its DNA synthesis activity more than fivefold in the presence or absence of proliferating cell nuclear antigen. Analysis of reaction products demonstrated the stimulation to be partly due to an increased processivity of pol {delta} but more importantly to an increase in its initiation frequency. Addition of ATP to reactions partially suppressed stimulation by WRNIP1. Furthermore, a mutant WRNIP1 lacking ATPase activity could stimulate pol {delta} normally but was insensitive to suppression by ATP. These results indicate that WRNIP1 functions as a modulator for initiation or restart events during pol {delta}-mediated DNA synthesis and that its ATPase activity is utilized to sense DNA ends and to regulate the extent of stimulation.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Biochemical dissection of DNA replication reactions in eukaryotes has revealed that they can be reconstituted with 10–20 components with various activities, most of which appear to be essential for cell proliferation (Waga & Stillman 1998; Bell & Dutta 2002). The sequential assembly of initiation factors at initiation origins allows the formation of replication fork complexes and subsequent processive progression on template DNA (Takisawa et al. 2000). However, recent studies of replication have hinted that the situation is considerably more complex (Sutton & Walker 2001). For example, factors required during postreplication DNA repair and for checkpoint responses are also involved in replication, since they are required not only to coordinate DNA synthesis and to maintain replicated chromosomal structures but also to respond to problems that arise during fork progression. These factors usually appear to be nonessential for cell proliferation since they are accessory or because they are redundant. However, an elucidation of their functions is indispensable to understanding the mechanisms that regulate fork progression in eukaryotic cells.

In the present study, we analysed the human Werner helicase interacting protein 1 (WRNIP1/Whip, Kawabe, et al. 2001), which is implicated in eukaryotic postreplication repair. WRNIP1 was originally identified as a mouse protein that physically interacts with a RecQ-like DNA helicase, Werner protein (WRN), mutation of which causes the genetic disorder Werner syndrome (Yu et al. 1996). Its budding yeast counterpart is MGS1 (maintenance of genome stability), which is involved in the maintenance of DNA topology and in postreplication DNA repair (Branzei et al. 2002a,b; Hishida et al. 2002). WRNIP1 is highly conserved from prokaryotes to eukaryotes and features a replication factor C (RFC)-like motif. It is also similar to the E. coli Holliday junction branch migration protein RuvB (Hishida et al. 2001; Branzei et al. 2002a). In budding yeast WRNIP1/Mgs1 has a tight functional interaction with DNA polymerase {delta} (pol {delta}). A null mgs1 mutation (mgs1{Delta}) partially suppresses growth defects conferred by mutations in POL3, POL31 or POL32, which encode the three subunits of pol {delta}, and MGS1 exhibits synthetic dosage lethality with at least two of these genes (Hishida et al. 2001; Branzei et al. 2002a). These results suggest that Mgs1 modulates the function of pol {delta} during replication or at some step in the postreplication repair pathway.

To elucidate the functions of WRNIP1 during replication, especially with respect to pol {delta}, we studied the biochemical characteristics of the human protein produced in baculovirus and E. coli expression systems. We found that human WRNIP1 (hWRNIP1) forms homo-oligomeric complexes that physically interact with human pol {delta} (hpol {delta}) and stimulate its DNA synthesis activity, mainly by increasing the frequency of initiation. These data provide the first biochemical evidence that WRNIP1 is involved in a eukaryotic replication fork complex and that it modulates pol {delta} activity.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
hWRNIP1 forms a homo-oligomeric complex

hWRNIP1 was purified on an anti-FLAG antibody-conjugated column from an insect-cell lysate expressing FLAG-hWRNIP1. We obtained near-homogenous hWRNIP1 by elution with FLAG-peptide (Fig. 1A). The eluate was further fractionated by Superose 6 gel filtration chromatography and glycerol gradient sedimentation (Fig. 1B,C). In these sizing steps, hWRNIP1 migrated as two components, one (major) of low molecular mass and the other (minor) of high molecular mass. The major peak corresponded to a sedimentation coefficient of 9.1 S and a Stokes radius of 160 ± 20 Å. Taking hydrodynamic irregularities into account, the native molecular mass was estimated to be 600 ± 75 kDa (Siegel & Monty 1966), about eight times higher than the monomer molecular mass of hWRNIP1, 73 kDa. This finding indicates that the protein exists in a homo-oligomeric complex, most likely an octamer. The minor peak of high molecular mass seen after both chromatographic steps likely reflects a regular assembly of the complex.



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  		Figure 1  Purification of the hWRNIP1 complex and measurement of activity. (A) Lanes Ly and Ft; insect cell lysate and the flow-through from an anti-FLAG column. Lanes 2–9; FLAG peptide eluted fractions from 2 to 9. Note that a protein band of about 99 kDa was detected for hWRNIP1. (B) Superose 6 (10/30) chromatography with 200 µL from FLAG-peptide elutate fraction 6. Aliquots (10 µL) were subjected to 10% SDS-PAGE and gels were stained with CBB. The elution position of thyroglobin (thy: Stokes radius of 160 Å) is indicated. Relative hWRNIP1 band intensities of the fractions are graphed below. (C) Glycerol gradient centrifugation of FLAG-peptide eluate fraction 6. After centrifugation, the sample was separated into 18 fractions from the bottom as indicated. Aliquots (10 µL) were treated as in (B). Sedimentation positions of catalase (11.3 S) and alcohol dehydrogenase (adh: 7.4 S) are indicated. The graph below the gel indicates relative values for hWRNIP1 band intensity (filled circles), ATPase activity determined with 0.5 µL each fraction (grey triangles) and fold stimulation of hpol {delta}-mediated DNA synthesis with 0.1 µL each fraction (grey boxes), as described in Experimental procedures. (D) ATPase activity was measured in 5 µL reaction mixtures with 90 ng hWRNIP1 (from fraction 8 in C) and the DNAs indicated on the right. HaeIII’ed pUC DNA, HaeIII-digested pUC118 DNA. The amount of Pi released from ATP after 20 min incubation at 37 °C is shown in pmol.

 
Since WRNIP1 shares amino acid sequence similarity with RFC, it may form a clamp-loader type complex, like the RFC family proteins Rad17 and Chl12 (Griffith et al. 2002; Shiomi et al. 2002; Ohta et al. 2002). To test this possibility, we coexpressed FLAG-hWRNIP1 with RFC small subunits (RFCs2–5) in insect cells. We previously reconstituted the novel clamp loader-type complexes Rad17/RFCs2-5 and Chl12/RFCs2-5 with the same method (Shiomi et al. 2002, 2004; Ohta et al. 2002). However, FLAG-hWRNIP1 expressed alone or with RFCs2-5 was isolated from an anti-FLAG column as a single component consisting of a homo-oligomeric complex (data not shown). Therefore, hWRNIP1 does not form a stable complex with RFCs2–5.

hWRNIP1 is an ATPase stimulated by DNA termini

hWRNIP1 has motifs typical of AAA+ family proteins (Ogura & Wilkinson 2001) and is expected to have ATPase activity. Indeed, this is the case for the S. cerevisae WRNIP1/Mgs1 protein stimulated with single- or double-stranded DNA (Hishida et al. 2001). When we assayed for ATPase activity in the glycerol gradient fractions, we found that it cosedimented with hWRNIP1 (Fig. 1C), indicating that hWRNIP1 has ATPase activity. We confirmed this observation by preparing a mutant protein, hWRNIP1(m), in which Thr294, a conserved residue in the nucleotide binding motif of AAA+ family proteins, was substituted with Ala. The purified hWRNIP1(m) exhibited almost no ATPase activity under the same conditions, although it was isolated as a similar homo-oligomeric complex (data not shown), indicating that the ATPase activity is intrinsic to hWRNIP1.

The DNA dependence of the ATPase activity was tested by adding increasing amounts of polydA, polydA/oligodT, pUC118 plasmid DNA or HaeIII-digested pUC118 (Fig. 1D). Interestingly, addition of polydA/oligodT or digested pUC118, but not of polydA or intact pUC118, efficiently stimulated activity, indicating a requirement for primer/template structures or double-stranded termini. However, further studies of hWRNIP1 interactions demonstrated no obvious binding to various types of DNA, suggesting that hWRNIP1 only weakly associates with DNA, if at all (data not shown).

hWRNIP1 binds to hpol {delta}

Previous genetic studies of budding yeast WRNIP1/Mgs1 demonstrated that it interacts genetically with two small pol {delta} subunits encoded by POL31 and POL32. Further analyses demonstrated that the mgs1{Delta} mutation can suppress the temperature- and hydroxyurea-sensitive phenotypes conferred by the cdc2-1 mutation, which affects a pol {delta} catalytic subunit (data not shown). These data indicate that WRNIP1 might have tight functional relations with pol {delta} (Hishida et al. 2001; Branzei et al. 2002a). To further explore this relationship, hpol {delta} purified from a baculovirus expression system was added to anti-FLAG antibody beads prebound to FLAG-hWRNIP1. As shown in Fig. 2A, the four subunits of hpol {delta} (125, 66, 50 and 12 kDa) were pulled down with FLAG-hWRNIP1 but not in its absence. To determine which subunits of hpol {delta} associate with hWRNIP1, each was expressed individually, and lysates were incubated with unbound anti-FLAG agarose beads or beads that had been prebound with FLAG-hWRNIP1. The cell lysates used contained almost equivalent amounts of components. We detected binding of the p125, p50 and p12 subunits but not the p66 subunit to the FLAG-hWRNIP1-bound beads (lane 9). Therefore, hWRNIP1 interacts with three of the four subunits of hpol {delta}.



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  		Figure 2  hWRNIP1 interacts with hpol {delta}. (A) anti-FLAG beads were preincubated with insect cell lysates expressing WRNIP1 (lanes 1, 2) or mock-infected lysates (lanes 3, 4). After extensive washing, the beads (lanes 1, 2, containing about 5 µg hWRNIP1) were mixed with purified pol {delta} (lanes 2, 4) or blank buffer (lanes 1, 3), and half of each bound fraction was immunoblotted with anti-pol {delta} p125, p66, p50 and p12 antibodies as indicated at left. Fifty ng of hpol {delta}, corresponding to one-sixth of the input, was used as a positive control. (B) hpol {delta} subunits were expressed individually and assayed for interaction with hWRNIP1 prebound to anti-FLAG-beads. Bound and unbound fractions from lysates containing almost equivalent amounts of subunits (lane 6) were subjected to immunoblot analysis with antibodies as indicated at left. A fivefold greater amount of ‘bound’ sample was loaded than of ‘input’ or ‘unbound’ samples.

 
We tested whether hWRNIP1 interacts with PCNA or RFC by the same method as shown in Fig. 2A. We observed a limited interaction with RFC but none with PCNA (data not shown). Since the amounts of the RFC recovered from the pull-down assay were much less than the amounts of hpol {delta}, these interactions were not studied further.

hWRNIP1 stimulates DNA synthesis activity

To determine the functional significance of these binding interactions, we added purified hWRNIP1 to a hpol {delta} DNA synthesis reaction mixture in the presence of PCNA (Fig. 3A). DNA synthesis mediated by hpol {delta} was significantly stimulated depending on the amount of added hWRNIP1. To conclusively determine whether hWRNIP1 is responsible for this stimulation activity, we assayed activity with fractions produced by glycerol gradient sedimentation (Fig. 1C) or by miniQ-anion exchange column chromatography (Fig. 3B). Peaks of activity were observed for fractions 4 and 8 of the glycerol gradient, exactly the same fractions that exhibited peaks of hWRNIP1 content and ATPase activity. A similar co-elution of hWRNIP1 and stimulation activity was observed for miniQ fractions 13–14, which were eluted at 0.33 M NaCl. These results indicate that the activity responsible for the stimulation of hpol {delta}-dependent DNA synthesis is tightly associated with hWRNIP1.



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  		Figure 3  Stimulation of hpol {delta} activity by hWRNIP1. PolydA/oligodT was used as a template for these experiments. (A) Specificity of stimulation. DNA synthesis was measured with increasing amounts of hWRNIP1 after incubation at 37 °C for 15 min in the presence of 0.5 ng hpol {delta} and 60 ng PCNA (open circles), 1 ng pol {delta} without PCNA (open triangles) or 1 ng DNA polymerase {alpha} (filled circles). Upper and lower panels indicate the extent of incorporation and fold stimulation of activity, respectively, and incorporation without hWRNIP1 was normalized to 1.0. (B) Co-elution of stimulation activity and hWRNIP1 in a miniQ column chromatography with 500 µL FLAG-peptide elutate fraction 6. Three µL aliquots were subjected to 10% SDS-PAGE and gels were stained with CBB. The graph below the gel indicates relative values for hWRNIP1 band intensity (lines with circles), fold stimulation of hpol {delta}-mediated DNA synthesis with 0.25 µL each fraction (shaded bars), and the NaCl gradient (broken line). (C) Comparison of hWRNIP1 activity produced in insect cells (open circles) or in E. coli (filled circles). Assays were done in the presence of 1 ng pol {delta}, 60 ng PCNA and the indicated amount of hWRNIP1. Both hWRNIP1 preparations were done by anti-FLAG and miniQ column chromatography.

 
WRNIP1 from mouse cells was detected as a protein that interacts with WRN helicase (Kawabe et al. 2001). Interestingly, human WRN helicase was also reported to interact with pol {delta} and to modulate its activity (Szekely et al. 2000; Kamath-Loeb et al. 2001). Thus, the stimulatory activity of our purified hWRNIP1 may have been due to a contaminating insect WRN that associated with the hWRNIP1 fraction. To rule out this possibility, we purified hWRNIP1 from the E. coli expression system and studied its activity. As indicated in Fig. 3C, the highly purified hWRNIP1 from E. coli also stimulated hpol {delta}.

Characterization of the stimulation of hpol {delta} by hWRNIP1

The extent of hpol {delta} stimulation was almost 10-fold with 20 ng hWRNIP1 under our experimental conditions and was hpol {delta}-specific, since we did not detect any appreciable stimulation of DNA synthesis by human DNA polymerase {alpha} (Fig. 3A). Furthermore, stimulation was independent of PCNA, since basal DNA synthesis mediated by hpol {delta} without PCNA was also stimulated to the same extent as synthesis in its presence (Fig. 3A, lower panel). We titrated the PCNA concentration in the hpol {delta} assay mixture with fixed amounts of hWRNIP1 and hpol {delta} and found that the maximal level of synthesis in the presence of hWRNIP1 was about twofold higher than in its absence (Fig. 4A).



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  		Figure 4  Characterization of pol {delta} stimulation by hWRNIP1 titration of PCNA for pol {delta}-mediated DNA synthesis with (filled boxes) or without (open boxes) 100 ng hWRNIP1. DNA synthesis was measured in 5 µL reaction mixtures with 0.75 ng hpol {delta} and the indicated amount of PCNA in the presence of 20 mM of NaCl after incubation at 37 °C for 15 min. (B) Titration of pol {delta} to assess stimulation with (open circles) or without (filled circles) 20 ng hWRNIP1. DNA synthesis was measured in 5 µL reaction mixtures with 60 ng PCNA and the indicated amount of hpol {delta} after incubation at 37 °C for 15 min. The fold stimulation of DNA synthesis is indicated by the broken line. (C) Effects of ATP and ATPase activity on the stimulation of hpol {delta} by hWRNIP1. hWRNIP1 (wild: filled circles and boxes) or hWRNIP1(m) (mutant: open circles and boxes) were titrated to measure pol {delta}-mediated DNA synthesis with (circles) or without (boxes) 1 mM ATP. DNA synthesis was measured in 5 µL reaction mixtures with 0.75 ng hpol {delta} and 60 ng PCNA at 37 °C for 15 min and the fold stimulation is indicated.

 
Next, we measured the extent of stimulation with varying concentrations of hpol {delta} with or without hWRNIP1. As indicated in Fig. 4B, only limited amounts of DNA synthesis occurred with 0.125–0.5 ng hpol {delta} in the presence of PCNA, probably due to the inefficient association of hpol {delta} with primer/template DNA. Under these conditions, substantial DNA synthesis (an up to 100-fold increase) was then observed upon the addition of hWRNIP1. In contrast, with 1 ng of hpol {delta}, which efficiently carried out DNA synthesis, the extent of stimulation by hWRNIP1 decreased. These data suggest that hWRNIP1 promotes the association of pol {delta} with primer/template DNA.

Stimulation and ATPase

Since hWRNIP1 has an ATPase activity stimulated by specific DNA structures, ATP might exert any on its pol {delta} stimulation, although we have observed stimulation without ATP in previous experiments. We studied the effects of ATP on stimulation as shown in Fig. 4C, and observed that the addition of 1 mM ATP to the reaction partially decreased stimulation. To determine the significance of the ATPase activity, we used an ATPase-deficient mutant, hWRNIP1(m), in the hpol {delta} stimulation assay. The purified hWRNIP1(m) was stimulatory with or without ATP, and the level was similar to that of wild-type hWRNIP1 in the absence of ATP (Fig. 4C), indicating that the ATPase activity is not required for the stimulation but that the active ATPase of hWRNIP1 exerts a negative influence on the stimulation of pol {delta} in the presence of ATP.

hWRNIP1 elevates the frequency of initiation events by hpol {delta}

Using polydA/oligdT as a template, the processivity of hpol {delta} was measured with or without hWRNIP1. Limited amounts of hpol {delta} were used to restrict incorporation to less than one nucleotide per primer end on average. The synthesized DNA products were purified and separated in a 2% alkaline agarose gel as shown in Fig. 5A. Smeared DNA products in the range of shorter than 100 to several thousand nucleotides long were detected. The products were slightly more elongated in the presence of hWRNIP1 but this result was not commensurate with a linear increase in incorporation with increasing amounts of hWRNIP1 (Fig. 5C). To quantify processivity, we measured the radioisotope intensity at each area in panel A and divided this value by the corresponding DNA length. The resulting value represents the relative abundance of the synthesized DNA strand (Fig. 5B). The graph illustrates the relative distribution of synthesized DNA strands from this calculation, taking the peak value as 1.0. The proportion of longer DNA strands synthesized by hpol {delta} became obviously higher upon the addition of hWRNIP1. If we calculated the processivity of hpol {delta} as an average length of the total synthesized DNA fragments, it was 670 nt under these experimental conditions without hWRNIP1, while it increased to 830 nt upon the addition of 20 ng hWRNIP1. An increase of hpol {delta} processivity was apparent but insufficient to explain the remarkable increase in incorporation. Therefore, stimulation of hpol {delta} by hWRNIP1 must be mainly caused by an increase in the initiation frequency.



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  		Figure 5  Analysis of products of hpol {delta}-mediated DNA synthesis stimulated by hWRNIP1. Five µL aliquots of reaction mixtures containing 250 pmol poly(dA)2300/oligo(dT)15, 80 ng hPCNA, 0.1 ng hpol {delta} and the indicated amount of hWRNIP1 (lanes 2–4: 5, 10, and 20 ng) were incubated at 37 °C for 15 min to measure DNA synthesis and carry out product analysis. (A) Autoradiography of products with roughly the same number of incorporated counts (except for lane 1 where about one third counts was loaded), after separation in a 2.0% alkaline agarose gel. Migration positions of single-stranded DNA size standards are indicated at left. (B) Relative abundance of synthesized DNA strands with or without 20 ng hWRNIP1 calculated by division of signal intensities at 50 base intervals with the corresponding DNA length using the peak value as 1.0. Processivity of hpol {delta} in this experiment was calculated as an average length of the total synthesized DNA fragments. (C) Comparison between the processivity of hpol {delta} and DNA synthesis activity with the indicated amounts of hWRNIP1 under the conditions described above. (D) Stimulation by hWRNIP1 in a pol {delta} holoenzyme assay. Five µL reaction mixtures containing 8 fmol singly primed mp18 viral DNA, 0.25 ng hpol {delta}, 15 ng PCNA, 28 ng RFC, 150 ng RPA and hWRNIP1 (lanes 2–4: 0, 50 100 ng) were incubated at 37 °C for 30 min. Reaction products were separated in a 1.2% alkaline agarose gel and visualized by autoradiography. Levels of DNA synthesis (pmol of TMP) are indicated at bottom. Lane 1 shows products synthesized without hpol {delta}. A fivefold longer exposure of lane 2 is shown in lane 5.

 
Differences in DNA synthesis modes of hpol {delta} with or without hWRNIP1 were studied with a native DNA template, singly primed single-stranded M13 phage DNA (Fig. 5D). The DNA synthesis reaction is dependent on RFC, PCNA and RPA, a eukaryotic single-stranded DNA binding protein. Under our conditions, hpol {delta} could synthesize only a limited amount of a full-length product, even after 30 min incubation (lane 2). The average length of synthesized DNA was less than 1 kb. However, a remarkable elongation of products was seen on the addition of hWRNIP1 (lanes 3–4). Taking the observed processivity of hpol {delta} on synthetic DNA into consideration, this result indicates that reinitiation of hpol {delta} elongation is promoted by hWRNIP1 on this native DNA template. However, we could not rule out the possibility that hWRNIP1 increases processivity under this particular condition. Interestingly, the distributions of apparent pause sites, which could be observed upon longer exposure, were not changed significantly by hWRNIP1 (lane 5). Therefore, hWRNIP1 may not change the tendency of hpol {delta} to pause on native DNA templates.


   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
The WRNIP1 complex

The present study clearly demonstrates that WRNIP1 predominantly forms a homo-octameric complex. Therefore, WRNIP1 is functionally distinguishable from other RFC-like proteins. In the predicted RFC structure based on the E. coli clamp loader protein {gamma} complex, the three small RFC subunits (RFCs2, 4, 5), located inside the pentameric alignment, have complete sets of ATPase motifs, whereas RFC1 and 3, possibly located at either end, have incomplete sets (O'Donnell et al. 2001). Two RFC1-like proteins, Rad17 and Chl12, which have incomplete ATPase motifs, can form clamp loader-type complexes by substituting for RFC1 (Dean et al. 1998; Mayer et al. 2001). In contrast, WRNIP1 has complete motifs and is much more similar to RFCs2, 4, 5, so that it has a structure different from that formed by other RFC1-like proteins. WRNIP1 also shares similarity with the E. coli RuvB protein, which forms a hexameric ring complex, exhibits ATPase activity and drives DNA strand migration at Holliday junction structures in the presence of the RuvA complex (Yamada et al. 2002). The functional involvement of WRNIP1 in Holliday junction migration in eukaryotes has not been reported.

Interaction of WRNIP1 with pol {delta}

As suggested by the genetic interaction of WRNIP1 with pol {delta} subunits in budding yeast, hWRNIP1 physically binds to hpol {delta} and interacts with several subunits (p125, p50 and p12). The lack of interaction with the p66 subunit, however, suggests specificity. pol {delta} also interacts with PCNA through both p125 and p66 via characteristic PCNA binding motifs, although the significance of binding to p66 remains unclear (Shikata et al. 2001). Our observations indicate that the two stimulatory factors PCNA and WRNIP1, both of which form homo-oligomeric complexes, also interact with pol {delta} via multiple contacts. Further studies are necessary to elucidate the significance of this feature, one possibility being the presence of a regulatory mechanism for stimulation through change in the numbers or combinations of contacts.

Relationship with the WRN helicase

It has been demonstrated that human WRN helicase interacts with pol {delta} (Szekely et al. 2000) and stimulates S. cerevisiae pol {delta} DNA synthesis activity (Kamath-Loeb et al. 2001). This stimulation depends on the third subunit of pol {delta} Pol32 (Kamath-Loeb et al. 2001), which corresponds to human p66. Human WRN also interacts with the other human pol {delta} subunit, p50 (Szekely et al. 2000). Thus, this protein has multiple association sites with pol {delta}. In turn, hWRNIP1 interacts with three of the pol {delta} subunits but not p66. This means that in addition to their common target p50, both proteins have independent target sites in the pol {delta} complex, suggesting that they can interact simultaneously with pol {delta}.

Interaction between WRN and WRNIP1 was first demonstrated by a yeast two-hybrid analysis of mouse cDNAs and by the genetic interaction of their S. cerevisiae homologues, Mgs1 and Sgs1 (Kawabe et al. 2001). Therefore, taking into account their functional interaction and their possible simultaneous associations with pol {delta}, these proteins are expected to form a ternary complex in functional situations (Fig. 6). Further studies are necessary to confirm the presence of this ternary complex and to elucidate the roles of the two regulatory factors during pol {delta} DNA synthesis.



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  		Figure 6  Model of a ternary complex containing WRNIP1, WRN and pol {delta} at an arrested replication fork. Based on previous reports and the present work, WRNIP1, WRN and pol {delta} may form a ternary complex. WRN and pol {delta} also interact with PCNA. This complex may function to regulate pol {delta}-mediated DNA synthesis when the replication fork complex is stalled by DNA damage or structural stress. The ATPase activity of WRNIP1 functions as a sensor of DNA ends, and ATP hydrolysis regulates the stimulation of pol {delta}. Thus, complex formation plays a crucial role in the re-initiation of stalled replication forks.

 
Functional significance of the stimulation of pol {delta} by WRNIP1

hWRNIP1 stimulates hpol {delta} specifically, independent of PCNA. Since the maximal level of DNA synthesis stimulated by PCNA was further increased by the addition of hWRNIP1, the mechanisms of stimulation mediated by the two proteins must be different. Indeed, hWRNIP1 did not increase the processivity of hpol {delta} in the absence of PCNA (data not shown), although hWRNIP1 stimulated DNA synthesis more than fivefold under these conditions. In the presence of PCNA, hWRNIP1 apparently increases the processivity of hpol {delta}. These results suggest that WRNIP1 behaves as a component of the pol {delta} complex as it migrates on a template DNA.

As indicated by product analyses, the main influence of WRNIP1 appears to be an increase in the initiation efficiency of pol {delta} DNA synthesis. Studies with lower amounts of pol {delta} also showed that WRNIP1 enhanced its recognition of primer DNA ends. Since WRNIP1/Mgs1 in budding yeast is not essential for cell growth, stimulation of pol {delta} by WRNIP1 is not critical for replication. Furthermore, although WRNIP1 clearly stimulates pol {delta}in vitro, the cellular level of WRNIP1/Mgs1 in yeast usually correlates inversely with pol {delta} functions (Hishida et al. 2001; Branzei et al. 2002a). This suggests that stimulation of pol {delta} by WRNIP1 might be detrimental for normal replication, possibly by disturbing the balance between leading and lagging DNA synthesis. Indeed, stimulation of hWRNIP1 carrying an active ATPase is suppressed by ATP, as representing its functional situation. Thus, WRNIP1 might be recruited to pol {delta} sites only under particular conditions, for example to an arrested replication fork complex. It is known that when a replication fork stops at a site of DNA damage, various mechanisms are activated in response, including translesion DNA synthesis or postreplicational repair. Stimulation of pol {delta} by WRNIP1 may be required during or after these processes. As we observed, double-stranded DNA ends or primer/template structure stimulated its ATPase activity. Thus, we assume that the ATPase activity functions as a sensor of DNA damage or of arrested replication forks, and that ATP hydrolysis induces a WRNIP1 function that stimulates initiation of pol {delta} DNA synthesis (Fig. 6). Indeed, a mutation affecting the ATPase domain of Mgs1 abolishes its genetic interaction with pol {delta}, indicating a crucial role of the ATPase for regulation of pol {delta} activity.

As suggested before, under normal conditions, WRN helicase might join a pol {delta}-WRNIP1 complex at the arrested replication fork and collaborate with WRNIP1 to allow pol {delta} to re-initiate DNA synthesis. Therefore, these two proteins play crucial roles in the maintenance of eukaryotic replication fork activity by modulating pol {delta} function according to local conditions, although they are not essential for DNA replication. Their actual roles should be tested in future by reconstitution with arrested replication fork DNA models and purified components.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Construction of WRNIP1 expression systems

The hWRNIP1 cDNA clone IMAGE: 4906400 was purchased from BD Biosciences Clontech (NJ, USA), and a synthetic oligonucleotide encoding the FLAG-tag sequence was inserted into a NcoI site at the initiation codon. The resulting DNA fragment was then inserted into the plasmid pBacPAK8, and recombinant baculovirus expressing FLAG-tagged hWRNIP1 was prepared using the BacPAK Baculovirus Expression System (BD). Preparation of viruses for expression of other proteins has been previously described (Shiomi et al. 2000; Shikata et al. 2001). Insect High 5 cell lysates infected with recombinant baculoviruses were prepared as described (Shiomi et al. 2000). The same DNA fragment was also inserted into pET28b (Novagen, Merck kgaA, Darmstadt, Germany) to obtain pET-WRNIP. This plasmid was propagated in the E. coli strain Rosetta (DE3) (Novagen) to produce E. coli-expressed WRNIP1.

The cDNA encoding a mutant hWRNIP1, hWRNIP1(m) was obtained by low-fidelity PCR and a FLAG-tagged derivative was inserted into pBacPAK8 to obtain the expression baculovirus (details will be described elsewhere).

Pull-down assay for FLAG-hWRNIP1

High 5 insect cell lysates expressing FLAG-hWRNIP1 were incubated with 10 µL anti-FLAG agarose beads (M2, Sigma) at 0 °C for 1 h. The beads were washed four times with 50 µL buffer H [25 mM HEPES-NaOH (pH 7.4), 1 mM ethylenediamine tetraacetic acid (EDTA), 10% glycerol, 0.5% Nonidet P-40, 1 mM phenyl methylsulphonyl fluoride and 2 µg/mL leupeptin) containing 0.1 M NaCl and incubated with 300 ng purified hpol {delta} in 10 µL buffer H containing 0.1 M NaCl at 0 °C for 1 h. The beads were washed three times with 100 µL buffer H containing 0.05 M NaCl and the bound proteins were eluted with 20 µL 10 mM glycine (pH 2.2). Half of each eluate was resolved by 15% SDS-PAGE, stained and immunoblotted as previously described (Shiomi et al. 2000; Shikata et al. 2001). Pull-down assays of individual pol {delta} subunits with hWRNIP1 were conducted as detailed above, except that 50 µL aliquots of insect cell lysates containing almost equivalent amounts of p125, p66, p50 or p12 were used instead of purified hpol {delta}.

Purification of FLAG-hWRNIP1

A cell lysate from 2 x 108 infected High 5 cells expressing the protein was prepared and fractionated on an anti-FLAG column (0.8 x 1.5 cm) as described (Shiomi et al. 2002). Near-homogeneous FLAG-hWRNIP1 was eluted with 4 mL buffer H containing 0.1 M NaCl and 100 µg/mL FLAG peptide. A 100 µL aliquot of the peak fraction was loaded onto a 2.2 mL glycerol gradient (15–35%) in buffer H (pH 7.5) containing 0.1 M NaCl and centrifuged at 50 000 r.p.m. in a TLS55 rotor (Beckman Instruments) for 14 h at 4 °C. Eighteen fractions of about 125 µL each were collected from the bottom of the tube. One hundred or 500 µL aliquots of the FLAG-peptide eluate were also fractionated on a Superose 6 column (HR10/30; Amersham Biosciences) in buffer H (pH 7.5) containing 0.15 M NaCl or on a miniQ column (PC 3.2/3, Amersham Biosciences) by elution with a 2 mL NaCl 0.1 M to 0.5 M gradient in buffer H.

To obtain E. coli-expressed hWRNIP1, a lysate prepared from a one-litre culture expressing FLAG-hWRNIP1 was also subjected to anti-FLAG and miniQ column chromatography as described above.

DNA synthesis assays

hpol {delta} was purified from insect cells infected with baculoviruses expressing the four subunits (Shikata et al. 2001 and details to be published elsewhere). Human DNA polymerase {alpha} complex was purified as previously described (Tsurimoto & Stillman 1991). DNA synthesis reactions were carried out in 5 µL reaction mixtures using poly(dA)400/oligo(dT)15 (Amersham Biosciences, 20 : 1) as a template (Tsurimoto & Stillman 1991). For reaction product analysis, poly(dA)2300 was prepared by extension of polydA (Amersham Biosciences) with terminal deoxynucleotide transferase to obtain poly(dA)2300/oligo(dT)15 (100 : 1 nucleotide ratio, 0.17 pmol of primer termini) for use as a primer/template. After DNA synthesis in the presence of [{alpha}-32P] dTTP (800 cpm/pmol), the product was precipitated with ethanol, dissolved in 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA and resolved on a 2.0% agarose gel under alkaline conditions (Tsurimoto & Stillman 1991). After autoradiography, the relative intensities of incorporated radioisotopes were determined using the Kodak 1D 3.5.2 program, and the relative number of DNA fragments was estimated by dividing intensity by the corresponding DNA length. Processivity was calculated as the average length of DNA fragments produced throughout the gel, as determined from the relative numbers.

For studies of DNA synthesis products with a native DNA template, a reaction mixture (20 µL) containing 30 mM HEPES NaOH (pH 7.6), 7 mM MgCl2, 0.5 mM DTT, 0.1 mg/mL bovine serum albumin, 25 µM each dATP, dCTP and dGTP and [{alpha}-32P]dTTP (800 cpm/pmol), 2 mM ATP, 8 fmol singly primed M13 DNA, 600 ng human replication protein A (RPA) (Tsurimoto & Stillman 1991), 100 ng FLAG-RFC (Shiomi et al. 2002), 60 ng PCNA (Fukuda et al. 1995) and 0.25 ng of hpol {delta}, as well as the indicated amount of FLAG-hWRNIP1, was incubated at 37 °C for the indicated time period, and samples of 4.5 µL each were collected. The reactions were stopped, radioactivity was determined, and replication products were analysed by 1.2% alkaline-agarose gel electrophoresis followed by autoradiography, as described above.


   Acknowledgements
 
We thank Dr Masukata (Osaka University, Japan) for his critical reading of this manuscript and valuable comments. The work described was supported by grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


   Footnotes
 
Communicated by: Hiroyuki Araki

aPresent address: Amersham Biosciences K.K. Sanken Bldg. 3-25-1 Hyakunincho, Shinjuku-ku, Tokyo 169–0073, Japan Back

* Correspondence: E-mail: ttsurscb{at}mbox.nc.kyushu-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 26 August 2004
Accepted: 14 October 2004




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