Lesion Bypass by the Escherichia coli DNA Polymerase V Requires Assembly of a RecA Nucleoprotein Filament*

Nina B. ReuvenDagger , Gali AradDagger , Alicja Z. Stasiak§, Andrzej Stasiak§, and Zvi LivnehDagger

From the Dagger  Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel and the § Laboratoire d'Analyse Ultrastructurale, Batiment de Biologie, Universite de Lausanne, CH-1015 Lausanne-Dorigny, Switzerland

Received for publication, July 31, 2000, and in revised form, November 13, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Translesion replication is carried out in Escherichia coli by the SOS-inducible DNA polymerase V (UmuC), an error-prone polymerase, which is specialized for replicating through lesions in DNA, leading to the formation of mutations. Lesion bypass by pol V requires the SOS-regulated proteins UmuD' and RecA and the single-strand DNA-binding protein (SSB). Using an in vitro assay system for translesion replication based on a gapped plasmid carrying a site-specific synthetic abasic site, we show that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. This is based on the reaction requirements for stoichiometric amounts of RecA and for single-stranded gaps longer than 100 nucleotides and on direct visualization of RecA-DNA filaments by electron microscopy. SSB is likely to facilitate the assembly of the RecA nucleoprotein filament; however, it has at least one additional role in lesion bypass. ATPgamma S, which is known to strongly increase binding of RecA to DNA, caused a drastic inhibition of pol V activity. Lesion bypass does not require stoichiometric binding of UmuD' along RecA filaments. In summary, the RecA nucleoprotein filament, previously known to be required for SOS induction and homologous recombination, is also a critical intermediate in translesion replication.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Genomic DNA is afflicted by numerous lesions that might interfere with its propagation and with gene expression (1). Most of these lesions, which are usually base modifications, are repaired by cellular DNA repair mechanisms (1). When the replication fork encounters a blocking DNA lesion that has escaped repair, replication stops forming a ssDNA1 region in DNA (2). In Escherichia coli at least two mechanisms, which are regulated by the SOS response (3), act to repair the gap by converting the ssDNA region into a dsDNA region without actually removing the damaged nucleotide. Recombinational repair patches the gap with a complementary DNA segment from the fully replicated sister chromatid (4, 5), whereas translesion replication fills in the gap by DNA synthesis. This pathway, also termed lesion bypass or error-prone repair, is mutagenic, because DNA lesions often cause misincorporation by DNA polymerases, leading to the formation of mutations (2, 6).

The in vitro reconstitution of SOS translesion replication with purified components (7-9) established that SOS-targeted mutagenesis occurs by replication through DNA lesions by DNA polymerase V (UmuC)2 in the presence of UmuD', RecA, and SSB (10, 11). Pol V effectively bypasses a synthetic abasic site (10, 11), a cyclobutyl TT dimer and a 6-4 TT adduct (12), leading to targeted mutations. When replicating an undamaged DNA template pol V is highly mutagenic and forms preferentially purine-purine and pyrimidine-pyrimidine mismatches, resulting in transversion mutations (13). These activities of pol V are responsible for SOS mutagenesis targeted to DNA lesions and for untargeted mutagenesis, which occurs in undamaged DNA regions.

Proteins similar to UmuC are widespread from E. coli to humans. Several of them were shown to encode DNA polymerases in bacteria (14), in Saccharomyces cerevisiae (15, 16), and in humans (reviewed in Refs. 17-19). Unlike pol V, these polymerases do not require any additional proteins for their polymerase and/or lesion bypass activities, at least in vitro. In this sense pol V is more similar to the recently discovered DNA polymerase RI, product of the plasmidic mucB gene, whose activity also requires RecA and SSB (20). Most intriguing is the requirement for RecA, a protein known to be required also for gap filling by recombinational repair. Here we show that the assembly of a RecA nucleoprotein filament is required for translesion synthesis by pol V.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Proteins-- UmuD' and MBP·UmuC (a fusion of UmuC to maltose-binding protein) were overexpressed and purified as previously described (8, 11). The degree of purity was estimated to be >95% for UmuD' and 90-95% for MBP·UmuC. SSB and RecA were purified as described (21, 22), except that a phosphocellulose purification step was added for RecA. Their degree of purity was estimated to be >95%. Restriction nucleases, T4 DNA ligase, and T4 polynucleotide kinase were from New England BioLabs; T7 gp6 exonuclease was from Amersham Pharmacia Biotech, and ATPgamma S was from Sigma.

DNA Substrates-- The preparation of the gapped plasmid GP21 carrying a site-specific lesion was recently described (23, 24). GP21 contains a site-specific synthetic (tetrahydrofuran) abasic site and a ssDNA region of ~350 nucleotides (see Fig. 1).

Translesion Replication Assay-- The translesion replication reaction was performed as previously described (8, 11) with minor changes. The standard reaction mixture (25 µl) contained 20 mM Tris-HCl, pH 7.5, 8 µg/ml bovine serum albumin, 5 mM dithiothreitol, 0.1 mM EDTA, 4% glycerol, 1 mM ATP, 10 mM MgCl2, 0.1 mM each of dATP, dGTP, dTTP, and dCTP, 0.1 µg (2 nM) of gapped plasmid, 50 nM SSB, 2 µM RecA, 500 nM UmuD', and 5-220 nM pol V (MBP·UmuC). Reactions were carried out at 37 °C as follows. RecA and the DNA substrate were incubated in the assay buffer for 2 min at 37 °C, and were then transferred to ice. Next SSB and UmuD' were added, and reactions were incubated for 4 additional min at 37 °C. MBP·UmuC was then added, and incubation continued for the indicated periods of time. Reactions were stopped by adding 20 mM EDTA, 0.2% SDS, and 100 mM NaCl (final concentrations) followed by heat inactivation (65 °C for 10 min), and the DNA was purified from the proteins by proteinase K digestion followed by phenol/chloroform extraction and ethanol precipitation. The purified DNA was digested with Asp700 (5 units) at 37 °C for 2 h. Then, 5 units of BstXI were added, and incubation was continued at 55 °C for another 2 h. This restriction yielded radiolabeled DNA fragments 15, 25, and 43 nucleotides long, representing the unextended primer, the replication stop at the lesion, and the bypass product, respectively. Alternatively, a modified procedure was used as follows. The purified DNA mixture was treated with calf intestine alkaline phosphatase (0.2 units, 1 h, 37 °C) to hydrolyze remaining dNTPs (11). The DNA was then digested with Asp700 (5 units) and MspA1I (5 units) at 37 °C instead of BstXI. This produced radiolabeled DNA bands which were 4 nucleotides longer than those obtained with the Asp700/BstXI cleavage, namely, fragments 19, 29, and 47 nucleotides long, representing the unextended primer, the replication stop at the lesion, and the bypass product, respectively (see Fig. 1). The DNA samples were fractionated by 15% PAGE-urea, followed by phosphorimager analysis (Fuji BAS 2500). The extent of bypass was calculated by dividing the amount of bypass products by the amount of the extended primers (lesion bypass). When indicated, the bypass was also calculated differently, by dividing the amount of bypass products by the total amount of DNA (molecules bypassed of total).

Electron Microscopy-- Protein-DNA complexes were fixed by addition of glutaraldehyde to 0.2% followed by a 15-min incubation at 37 °C (complexes stabilized with ATPgamma S did not require fixation). Samples were diluted and washed in 5 mM magnesium acetate before uranyl acetate staining, as previously described (25). Complexes were visualized at magnifications of × 20,500 using a Philips CM100 electron microscope.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

RecA Is Needed in Stochiometric Amounts for Lesion Bypass-- The experimental system for assaying lesion bypass was previously described (8, 23). It is based on a gapped plasmid carrying a site-specific synthetic abasic site in the ssDNA region and an internal radiolabeled phosphate in the primer strand (Fig. 1). Upon addition of pol V the 3'-primer terminus is extended up to the abasic site, where synthesis may terminate or proceed through the abasic site (lesion bypass). At the end of the reaction the DNA is isolated and restricted with MspA1I, which cleaves 4 nucleotides upstream of the radiolabel, and with Asp700, which cleaves downstream of the lesion. This yields radiolabeled DNA fragments of 19, 29, and 47 nucleotides long, for the unextended primer, the block at the lesion, and the bypass product, respectively (Fig. 1). Bands in the length range of 20-46 nucleotides represent pol V pause or dissociation sites (see for example Figs. 2 and 6). In some cases the DNA products were restricted with Asp700 and BstXI, yielding products which were 4 nucleotides shorter than with the Asp700/MspA1I cleavage. The DNA were fractionated by urea-PAGE and visualized and quantified by phosphorimaging.



View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1.   Outline of the translesion replication assay using a gap-lesion plasmid. The gap-lesion plasmid substrate is represented above with restriction sites as indicated. Products of the lesion bypass assay following cleavage by restriction nucleases are represented below. Filled rectangle, synthetic abasic site; filled circle, radiolabeled phosphate. See text for details.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of RecA concentration on lesion bypass by pol V. Reactions were performed with the gap-lesion plasmid GP21, 100 nM pol V (as MBP·UmuC), 1.25 µM UmuD'2, 600 nM SSB tetramers, and the indicated concentrations of RecA, for 4 min at 37 °C. Restriction of the DNA products prior to PAGE analysis was done with Asp700 and MspA1I. The details are described under "Materials and Methods." Upper panel, phosphorimage of the gel. Lower panel, quantification of the results shown in the phosphorimage.

The activity of the RecA protein in recombination or SOS induction requires its binding to DNA. This binding is stoichiometric and cooperative, leading to the formation of a helical RecA filament along ssDNA, composed of multiple RecA monomers (reviewed in Refs. 26, 27). We examined whether the formation of a RecA nucleoprotein filament is required also for lesion bypass. First, a titration experiment was performed, to determine the amount of RecA needed to saturate the bypass reaction. As can be seen in Fig. 2, although lesion bypass could be observed at 0.5 µM RecA, 2 µM RecA was required to reach a saturation level of bypass. Further increases up to 16 µM RecA had little effect on lesion bypass (Fig. 2). For comparison, a titration of pol V was performed. As can be seen in Fig. 3, bypass activity reached saturation at 50-100 nM pol V, 20-40-fold lower than the saturating concentration of RecA. The concentration of the DNA in the reaction was 2 nM of gapped molecules, which translates into 700 nM nucleotides in the ssDNA region. Because a single RecA molecule binds 3-4 nucleotides (26), this means that ~200 nM RecA will be sufficient to cover the ssDNA region. The result that the saturating concentration of RecA is 10-fold higher than required to cover the ssDNA region is consistent with the formation of RecA nucleoprotein filament, reflecting the equilibrium between bound and free RecA. In addition, the RecA filament may extend to the double-stranded portion of the DNA molecule, as previously shown (28-30).



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of pol V concentration on lesion bypass. Reactions were performed as described in the legend to Fig. 2, except that RecA was at 4 µM, MBP·UmuC was at the indicated concentrations, and reaction time was 8 min. Restriction of the DNA products prior to PAGE analysis was done with Asp700 and BstXI. The details are described under "Materials and Methods." Upper panel, phosphorimage of the gel. Lower panel, quantification of the results shown in the phosphorimage.

Lesion Bypass Requires Single-stranded Gaps That Are Longer than 100 Nucleotides-- The formation of a stable RecA nucleoprotein filament requires a ssDNA region longer than 50 nucleotides (26). Thus, if a stable RecA nucleoprotein filament is required for lesion bypass, this should be reflected in a requirement for a long ssDNA region in the substrate DNA. This prediction was examined by using DNA substrates with single-stranded regions 12, 100, 350, or 850 nucleotides long. Fig. 4 shows the results of lesion bypass with DNA gaps 12 and 350 nucleotides long. In these experiments, the primer terminus was located opposite the nucleotide preceding the lesion in the template strand. This configuration of the DNA substrate gave lesion bypass results similar to those obtained with the standard substrate where the primer terminus was located 11 nucleotides upstream of the lesion (data not shown). As can be seen in Fig. 4, when the DNA substrate contained a small gap of 12 nucleotides, no bypass was observed by pol V, UmuD', RecA, and SSB (Fig. 4, lane 7). Furthermore, in a control reaction, bypass by pol I was higher than pol V on this substrate (Fig. 4, lane 2 versus lane 7). Attempts to examine whether omission of one of the protein components will allow lesion bypass on this DNA substrate showed no bypass under all the conditions examined (Fig. 4, lanes 3-6). No bypass was observed even with a 100-nucleotide-long gap (data not shown). In contrast, effective bypass was observed with gaps of 350 (Fig. 4, lane 14; see also Figs. 2 and 3) or 850 nucleotides long (see Fig. 8 below).



View larger version (71K):
[in this window]
[in a new window]
 
Fig. 4.   Pol V is unable to perform translesion synthesis on a substrate with a 12-nucleotide gap. The gapped plasmid substrates contain a primer ending one nucleotide before the lesion and gaps of 12 or 350 nucleotides long. Reactions were performed with the indicated combinations of proteins for 8 min. The concentrations of the proteins were: MBP·UmuC, 220 nM; UmuD'2, 1.25 µM; RecA, 4 µM; SSB, 600 nM (as tetramers); and pol I, 90 nM. Restriction of the DNA products prior to PAGE analysis was done with Asp700 and BstXI. The details are described under "Materials and Methods." The phosphorimage of the gel is presented.

RecA Nucleoprotein Filaments Are Formed under Bypass Reaction Conditions-- The formation of RecA nucleoprotein filaments under the conditions of the bypass reaction was examined using electron microscopy. The components of the reaction were mixed and incubated at 37 °C. Then the mixture was treated with glutaraldehyde to form protein-protein and protein-DNA cross-links and visualized by electron microscopy. As can be seen in Fig. 5C, RecA formed a filament along the ssDNA region in the DNA substrate. The length of the filament corresponds to the size of the gap in the plasmid. A similar picture was obtained when both RecA and SSB were present (Fig. 5D) or when the complete bypass reaction mixture was analyzed (Fig. 5F), although the size of the protein filament is somewhat larger. This may reflect coverage of the dsDNA adjacent to the gap; however, binding of RecA was largely restricted to the ssDNA region. This is not because of a shortage in RecA, because upon addition of ATPgamma S, all DNA molecules were entirely covered with RecA (Fig. 5E).



View larger version (210K):
[in this window]
[in a new window]
 
Fig. 5.   Electron microscopy analysis of complexes formed between the gap-lesion plasmid and protein components of the translesion replication reaction. A, the gap-lesion plasmid GP21. B, GP21 incubated with 50 nM (as tetramers) SSB. C, GP21 incubated with 2 µM RecA. D, GP21 incubated with 2 µM RecA and 50 nM SSB. E, same as D but with the addition of ATPgamma S. F, GP21 incubated under standard bypass conditions with 100 nM pol V, 500 nM UmuD'2, 2 µM RecA, and 50 nM SSB. Samples shown in A-D and F were treated after incubation with glutaraldehyde, whereas the sample shown in E was fixed with ATPgamma S, without glutaraldehyde cross-linking. The bar represents 100 nm.

ATPgamma S Inhibits Lesion Bypass-- ATPgamma S is a nonhydrolyzable ATP analog, which is known to strongly stabilize RecA nucleoprotein filaments (26). When lesion bypass was examined with ATPgamma S instead of ATP, there was a strong reduction in lesion bypass (Fig. 6, lanes 5-7). This effect of ATPgamma S was also observed in the presence of ATP (Fig. 6, lanes 11-13). Lesion bypass was calculated in this case by two methods. 1) The usual measure of lesion bypass was calculated as the percentage of bypass products of the primers that were extended. This measures lesion bypass directly, correcting for the efficiency of initiation of synthesis at the primer terminus. Molecules bypassed of total is the percentage of molecules on which bypass has occurred. For a substrate in which the primer terminus is located several nucleotides upstream to the lesion, this will be lower than lesion bypass, because usually not all primers are extended. As can be see in Fig. 6, the effect of ATPgamma S is primarily on the initiation of DNA synthesis, as indicated by the decrease in the fraction of primers that were extended, and by the molecules bypassed of total (Fig. 6, lanes 2-4 versus lanes 5-7). Additionally, it seems that the bypass step itself is also inhibited, but the effect is considerably smaller (Fig. 6; lesion bypassed, %). In the presence of ATPgamma S, the RecA filaments extend to cover the entire DNA molecule under our reaction conditions (see Fig. 5E, below). Therefore, the inhibition of pol V by ATPgamma S can be because of reduced accessibility to the primer terminus caused by the tight RecA·DNA complex. Alternatively, the altered conformation of the RecA nucleoprotein in the presence of ATPgamma S may be inhibitory. Lesion bypass was also observed in the absence of either ATP or ATPgamma S (Fig. 6, lanes 8-10), presumably because of the presence of dATP. This is consistent with previous results showing that dATP can replace ATP as a cofactor for RecA activities (26).



View larger version (69K):
[in this window]
[in a new window]
 
Fig. 6.   Translesion replication by pol V in the presence of ATPgamma S. Reactions were performed with the gap-lesion GP21, 90 nM pol V, 500 nM UmuD', 50 nM SSB (as tetramers), 2 µM RecA, and 1 mM ATP or ATPgamma S for 2, 4, and 6 min. In lanes 11-13, ATPgamma S was added from the beginning of the RecA·DNA preincubation, and ATP was added with the addition of pol V. Restriction of the DNA products prior to PAGE analysis was done with Asp700 and MspA1I. The details are described under "Materials and Methods." Bypass activity was calculated in two ways: 1)as a percentage of the extended primers (lesion bypass) or 2) as a percentage of total substrate molecules (molecules bypassed out of total). The value for percentage of primers initiated (of total substrate molecules) is also presented.

Requirement for SSB in Lesion Bypass-- SSB was previously shown to stimulate RecA-catalyzed reactions by facilitating the formation of a RecA nucleoprotein filament (31-33). This occurs at low SSB concentrations, which favor a limited cooperativity mode of binding to DNA, leading to the appearance of beads (octamers of SSB) on a string (ssDNA Ref. 33). To examine whether the SSB requirement in lesion bypass is related to the loading of RecA on DNA, a titration of SSB was performed. As can be seen in Fig. 7, the highest bypass activity was observed at 50 nM SSB. Increasing SSB concentration above 50 nM caused a slight decrease in bypass activity, followed by a plateau. Electron microscopy analysis of SSB binding to the gapped plasmid under our reaction conditions (50 nM SSB) revealed the appearance of beads on a string (see Fig. 5B, above). This is consistent with the notion that under lesion bypass reaction conditions, SSB binds ssDNA in the limited cooperativity mode and may facilitate the formation of a RecA nucleoprotein filament.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 7.   Effect of SSB concentration on lesion bypass by pol V. Reactions were performed with the gap-lesion plasmid GP21, 100 nM pol V, 1.25 µM UmuD', 4 µM RecA, and the indicated concentrations of SSB for 4 min. The graph shows the results of the translesion replication assay.

If the only function of SSB in bypass is to help assemble the RecA nucleoprotein filament, then bypass should be observed also in the absence of SSB. Similar situations exist for other RecA-catalyzed reactions such as strand exchange. This reaction is stimulated by SSB, but it occurs also in its absence (26). When SSB is absent, however, the concentration of Mg2+ should be kept low to prevent secondary structures from interfering with RecA polymerization on DNA (34). We have therefore performed a preincubation step of RecA and the DNA, in the absence of SSB, under 1 mM Mg2+. Then pol V and UmuD' were added, and the concentration of Mg2+ was raised to 10 mM to enable optimal activity of the polymerase. No bypass was observed under these conditions (data not shown). Other experimental protocols that were carried out included prolonged preincubation times with RecA prior to the addition of pol V, usage of higher concentrations of RecA, and addition of ATPgamma S instead of ATP. Under any of these conditions no bypass was observed in the absence of SSB (data not shown). In an attempt to examine whether a RecA nucleoprotein filament was formed in the absence of SSB, we assayed strand exchange between the gapped plasmid and a homologous DNA fragment. We found that RecA promoted strand exchange in the absence of SSB under the same conditions in which no lesion bypass was observed.3 This indicates that a functional RecA nucleoprotein filament was formed in the absence of SSB. Therefore, it seems that facilitating the formation of a RecA nucleoprotein filament is not the only role for SSB in lesion bypass.

Lesion Bypass Does Not Require Stochiometric Coverage of the RecA Nucleoprotein Filament by UmuD' or UmuD'C-- Plasmon surface resonance analysis has shown that complexes of UmuD'2C bind along a RecA nucleoprotein filament (35). This binding is mediated most likely through UmuD', which was previously shown to interact with RecA (36). So far, there was no demonstration of a direct binding between UmuC and RecA. A report that UmuC was retained on a column of immobilized activated RecA was based on a cell extract as a source of UmuC, and therefore binding could have been mediated via a third protein (37). The binding of UmuD'2C along the RecA nucleoprotein filament may be related to the tendency of multiple UmuD'2 molecules to form long filaments in crystalline state (38). This property of UmuD' might be also reflected in its structure in solution (39). To examine whether such filaments might be required for lesion bypass, the following experiments were performed. In addition to the standard substrate with a gap of 350 nucleotides, a DNA substrate with an extended gap of 850 nucleotides was also constructed. A stoichiometric binding of UmuD'2 or UmuD'2C along the RecA·ssDNA complex would be reflected in a requirement for higher concentrations of these proteins in the case of the extra large gap. We performed a titration of UmuD' under a constant concentration of 100 nM UmuC. As can be seen in Fig. 8, maximal bypass on the 350 nucleotides gap was obtained with 100 nM UmuD'2. This concentration is equimolar with the concentration of UmuC on the one hand, but it is also sufficient to cover the RecA-bound ssDNA region, assuming that one UmuD'2 molecule binds two RecA monomers (Ref. 35; As discussed above, there is 200 nM RecA bound to the ssDNA region). The UmuD' titration was repeated with a gap of 850 nucleotides, in the presence of a 3-fold higher RecA concentration (because of the larger size of the gap). It was found that the saturating amount of UmuD'2 was the same, 100 nM (Fig. 8), significantly less than the amount required to cover the RecA nucleoprotein filament (at least 240 nM). Moreover, the extent of maximal bypass was similar with both DNA substrates (Fig. 8). This suggests that under our assay conditions, binding of either UmuD'2 or UmuD'2C along the RecA nucleoprotein filament is not required for lesion bypass.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 8.   Titration of UmuD' required to saturate lesion bypass on DNA with large gaps. Reactions were performed on gap-lesion plasmids with a 350-nucleotide gap (GP21) or a 850-nucleotide gap (GP21XL) with 100 nM pol V and the indicated concentrations of UmuD' for 8 min. For GP21, 2 µM RecA and 50 nM SSB were added, whereas for GP21XL 6 µM RecA and 150 nM SSB were added. , GP21XL; , GP21.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Two of the most striking features of the activity of pol V are the ease with which it bypasses lesions that severely block other DNA polymerases and the absolute requirement for RecA (8-12). As discussed before, RecA is known to be required for another DNA damage tolerance mechanism, namely recombinational repair. Numerous studies have documented the binding of RecA to DNA in the form of a long helical nucleoprotein filament, with RecA wrapped around the DNA (reviewed in Refs. 27, 40). The following results argue that the assembly of a RecA nucleoprotein filament is required for lesion bypass by pol V. 1) RecA is required at stoichiometric concentrations in the lesion bypass reaction. 2) Electron microscopy analysis showed the existence of RecA nucleoprotein filaments under our assay conditions. 3) No bypass was found with DNA substrates in which the lesion was located on a ssDNA 100 nucleotides or shorter, on which a stable RecA filament is difficult to assemble.

On gapped DNA, the assembly of the RecA nucleoprotein filament starts in the ssDNA region, and proceeds in the 5'right-arrow3' direction (41). It can then continue to assemble on the double-stranded portion of the DNA (28-30). Based on the amounts of DNA and RecA, there is enough RecA to coat the DNA entirely. This is indeed seen in the electron micrograph in the presence of ATPgamma S (Fig. 5E). However, under standard reaction conditions, the ssDNA region is covered with RecA whereas the dsDNA region remains largely uncoated. This is not because of the presence of UmuC and UmuD', because the same results were obtained in their absence. Glutaraldehyde effectively cross-links proteins to proteins and to ssDNA, but not to dsDNA. However, glutaraldehyde treatment does fix the RecA filament on dsDNA, probably because of the special structure of the protein-DNA complex (42). Thus, it seems that the electron micrograph results showing the dsDNA largely uncoated, reflect the situation in solution under reaction conditions.

In the presence of ATPgamma S, where the entire DNA molecule was covered with RecA, there was a strong inhibition of the activity of pol V, primarily because of a 10-fold reduction in initiation of synthesis. This inhibition of initiation by pol V may have been caused by the tight and extended binding of RecA to DNA in the presence of ATPgamma S, leading to limited accessibility of pol V to the primer terminus. In addition, the inhibition may have been caused, at least in part, by the altered conformation of RecA filaments in the presence of ATPgamma S. The ATPgamma S experiments provide some hints on the dynamics of RecA during the action of pol V. A priori it is possible that the dissociation of RecA takes place only after the polymerization/bypass step. RecA stretches and unwinds the DNA and upon this the ssDNA in the RecA nucleoprotein filament is partially exposed (4, 5). Thus, the access of the polymerase to ssDNA might not be hindered in the presence of RecA. Nevertheless, the inhibitory effect of ATPgamma S on bypass is consistent with a model in which polymerization requires dissociation of RecA from DNA (Fig. 6).

SSB is known to facilitate the formation of RecA nucleoprotein filaments by removing secondary structures from DNA (27, 40). This occurs in the beaded limited cooperativity mode of binding of SSB to DNA. In this mode, octamers of SSB bind ssDNA forming a beaded structure, representing a binding mode of 65 nucleotides per SSB octamer (43, 44). Such binding allows entry sites for RecA, which then forms filaments via cooperative binding, with the displacement of SSB. Electron micrograph analysis under our reaction conditions revealed the appearance of complexes (Fig. 5B), which were consistent with the beaded structure of SSB·DNA complexes reported by Griffith (43). This supports a role for SSB in facilitating the formation of the RecA nucleoprotein filament required for lesion bypass. We were unable to observe lesion bypass in the absence of SSB. This means one of two possibilities, which are not mutually exclusive. 1) The RecA nucleoprotein filament in the presence of SSB is different from the filament of RecA alone, or 2) SSB has an essential role other than the loading of RecA on DNA. It is possible that mixed RecA·SSB complexes with DNA are required for lesion bypass. Such mixed complexes were previously described (45).

The importance of the proper assembly of a RecA nucleoprotein filament is likely to be the reason for the difference between our results and those of Goodman and co-workers (10). Whereas in our system only UmuC, UmuD', RecA and SSB were required for lesion bypass (11), in their system the processivity subunits of DNA polymerase III holoenzyme (the beta -subunit and the five subunit gamma  complex) were also required (10). This is not likely to be caused by a contamination of the 6 proteins in our protein preparations, which were highly purified. The extra requirements stem, in our view, from the difficulty in assembling a stable and functional RecA nucleoprotein filament on the particular substrate used by Goodman and his co-workers (10). They used a linear DNA substrate, with the lesion located on a single-stranded region, 50 nucleotides from the 5'-end of the DNA. Because RecA filaments assemble on DNA in a polar 5'right-arrow3' direction (41), this relatively short stretch of ssDNA 5' to the lesion may be incompletely covered with RecA, or else the complex may be unstable at the primer terminus. Addition of the processivity proteins presumably stabilized the protein-DNA complex, enabling bypass. This interpretation is supported by their observation that the requirement for processivity proteins was alleviated when ATPgamma S was used instead of ATP (12). In that case, the presence of ATPgamma S presumably promoted formation of a stable RecA filament, which could enable bypass to a low extent. Nevertheless, whereas not absolutely required for lesion bypass, the processivity proteins might have a stimulatory effect on pol V.

The inhibitory effect of ATPgamma S on bypass described above seems to contradict the report of Goodman and co-workers (12), who found that ATPgamma S stimulated bypass by pol V. This difference stems, most likely, from the difference in the absolute extents of bypass in the two systems. Goodman and co-workers (12) report a stimulation of bypass from a state of no bypass to low bypass, whereas we observed an inhibition from high bypass to low bypass (Fig. 6). Because no quantification was given in their paper for bypass in the presence of ATPgamma S, it is difficult to make a precise comparison. However, in both laboratories the bypass in the presence of ATPgamma S was low.

The RecA nucleoprotein filament is a tool used by the cell to protect the integrity of its DNA when replication is blocked at DNA lesions and single-stranded regions are exposed. Coating of the ssDNA with RecA protects it from degradation on the one hand (46) and provides the platform for repairing the gap by recombinational repair and translesion synthesis, on the other hand. Having all these activities require a common intermediate, i.e. the RecA nucleoprotein filament, offers an obvious effective mode of regulation and coordination of these processes. The relationship between recombinational repair and translesion synthesis is not clear. The two mechanisms might compete with each other or there might be a switching mechanism that governs the activities of the two pathways. In this context it should be mentioned that pol V itself was proposed to switch the cellular repair systems from recombination to bypass, by inhibiting recombinational repair (47, 48).

Based on current knowledge, the following model can be drawn for translesion replication (Fig. 9): When a replication fork encounters a blocking lesion, the DNA polymerase stops. At that stage the RecA protein displaces SSB from DNA, forming a RecA nucleoprotein filament. This nucleoprotein filament serves as a substrate for DNA polymerase V. The interaction between pol V and RecA is mediated most likely by UmuD'2, which interacts both with RecA and pol V. Pol V then replicates across the damaged site, incorporating a limited number of nucleotides. Because replication of undamaged regions of the DNA is both inefficient (because of the low processivity of pol V) and mutagenic, there is a switch back to error-free and efficient replication by DNA polymerase III holoenzyme. Obviously, this model provides only a preliminary scenario for translesion replication by pol V. Further studies are needed to elucidate the detailed mechanism of action of this fascinating DNA polymerase.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 9.   A model for translesion replication by pol V. The gray rectangle represents the DNA lesion, and M is an incorrect nucleotide inserted opposite the lesion by pol V. See text for details.



    FOOTNOTES

* This research was supported by Grants 96-00448 and 1999141 from the United States-Israel Binational Science Foundation. Grant 31-58841.99 from the Swiss National Science Foundation, and a grant from the Human Science Frontiers Program (to A. S. and A. Z. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Incumbent of The Maxwell Ellis Professorial Chair in Biomedical Research. To whom all correspondence should be addressed: Dept. of Biological Chemistry, Weizmann Inst. of Science, Rehovot 76100, Israel. Tel.: 972-8-934-3203; Fax: 972-8-934-4169; E-mail: zvi.livneh@weizmann.ac.il.

Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M006828200

2 We define UmuC as pol V, whereas Goodman and co-workers (10, 12) define the UmuD'2C as pol V. Until the function of UmuD' is elucidated, we prefer to avoid including UmuD' as part of the polymerase. Notice that UmuD' has no effect on the intrinsic polymerase activity of UmuC (in the absence of RecA and SSB, Ref. 11).

3 A. Berdichevsky and Z. Livneh, unpublished data.


    ABBREVIATIONS

The abbreviations used are: ssDNA, single-stranded DNA; SSB, single-strand DNA-binding protein; MBP, maltose-binding protein; ATPgamma S, adenosine 5'-O-thiotriphosphate; PAGE, polyacrylamide gel electrophoresis.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


1. Friedberg, E. C., Walker, G. C., and Siede, W. (1995) DNA Repair and Mutagenesis , ASM Press, Washington, D. C.
2. Livneh, Z., Cohen-Fix, O., Skaliter, R., and Elizur, T. (1993) Crit. Rev. Biochem. Mol. Biol. 28, 465-513[Abstract]
3. Little, J. W., and Mount, D. W. (1982) Cell 29, 11-22[Medline] [Order article via Infotrieve]
4. Eggleston, A. K., and West, S. C. (1996) Trends Genet. 12, 20-26[CrossRef][Medline] [Order article via Infotrieve]
5. Cox, M. M. (1998) Genes Cells 3, 65-78[Abstract/Free Full Text]
6. Walker, G. C. (1995) Trends Biochem. Sci. 20, 416-420[CrossRef][Medline] [Order article via Infotrieve]
7. Rajagopalan, M., Lu, C., Woodgate, R., O'Donnell, M., Goodman, M., and Echols, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10777-10781[Abstract]
8. Reuven, N. B., Tomer, G., and Livneh, Z. (1998) Mol. Cell 2, 191-199[Medline] [Order article via Infotrieve]
9. Tang, M., Bruck, I., Eritja, R., Turner, J., Frank, E. G., Woodgate, R., O'Donnell, M., and Goodman, M. F. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9755-9760[Abstract/Free Full Text]
10. Tang, M., Shen, X., Frank, E. G., O'Donnell, M., Woodgate, R., and Goodman, M. F. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8919-8924[Abstract/Free Full Text]
11. Reuven, N. B., Arad, G., Maor-Shoshani, A., and Livneh, Z. (1999) J. Biol. Chem. 274, 31763-31766[Abstract/Free Full Text]
12. Tang, M., Pham, P., Shen, X., Taylor, J. S., O'Donnell, M., Woodgate, R., and Goodman, M. F. (2000) Nature 404, 1014-1018[CrossRef][Medline] [Order article via Infotrieve]
13. Maor-Shoshani, A., Reuven, N. B., Tomer, G., and Livneh, Z. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 565-570[Abstract/Free Full Text]
14. Wagner, J., Gruz, P., Kim, S. R., Yamada, M., Matsui, K., Fuchs, R. P. P., and Nohmi, T. (1999) Mol. Cell 4, 281-286[Medline] [Order article via Infotrieve]
15. Nelson, J. R., Lawrence, C. W., and Hinkle, D. C. (1996) Nature 382, 729-731[CrossRef][Medline] [Order article via Infotrieve]
16. Johnson, R. E., Prakash, S., and Prakash, L. (1999) Science 283, 1001-1004[Abstract/Free Full Text]
17. Johnson, R. E., Washington, M. T., Prakash, S., and Prakash, L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 12224-12226[Free Full Text]
18. Woodgate, R. (1999) Genes Dev. 13, 2191-2195[Free Full Text]
19. Friedberg, E. C., Feaver, W. J., and Gerlach, V. L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5681-5683[Free Full Text]
20. Goldsmith, M., Sarov-Blat, L., and Livneh, Z. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11227-11231[Abstract/Free Full Text]
21. Lohman, T. M., and Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603[Abstract]
22. Cox, M. M., McEntee, K., and Lehman, I. R. (1981) J. Biol. Chem. 256, 4676-4678[Abstract/Free Full Text]
23. Tomer, G., Reuven, N. B., and Livneh, Z. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 14106-14111[Abstract/Free Full Text]
24. Tomer, G., and Livneh, Z. (1999) Biochemistry 38, 5948-5958[CrossRef][Medline] [Order article via Infotrieve]
25. Stasiak, A. Z., Larquet, E., Stasiak, A., Muller, S., Engel, A., Van Dyck, E., West, S. C., and Egelman, E. H. (2000) Curr. Biol. 10, 337-340[CrossRef][Medline] [Order article via Infotrieve]
26. Roca, A. I., and Cox, M. M. (1990) Crit. Rev. Biochem. Mol. Biol. 25, 415-456[Medline] [Order article via Infotrieve]
27. Kowalczykowski, S. C., Dixon, D. A., Eggleston, A. K., Lauder, S. D., and Rehrauer, W. M. (1994) Microbiol. Rev. 58, 401-465[Abstract]
28. Shaner, S. L., and Radding, C. M. (1987) J. Biol. Chem. 262, 9211-9219[Abstract/Free Full Text]
29. Shaner, S. L., Flory, J., and Radding, C. M. (1987) J. Biol. Chem. 262, 9220-9230[Abstract/Free Full Text]
30. Lindsley, J. E., and Cox, M. M. (1989) J. Mol. Biol. 205, 695-711[Medline] [Order article via Infotrieve]
31. Flory, J., and Radding, C. M. (1982) Cell 28, 747-756[Medline] [Order article via Infotrieve]
32. Cox, M. M., Soltis, D. A., Livneh, Z., and Lehman, I. R. (1983) J. Biol. Chem. 258, 2577-2585[Abstract/Free Full Text]
33. Kowalczykowski, S. C., and Krupp, R. A. (1987) J. Mol. Biol. 193, 97-113[Medline] [Order article via Infotrieve]
34. Tsang, S. S., Muniyappa, K., Azhderian, E., Gonda, D. K., Radding, C. M., Flory, J., and Chase, J. W. (1985) J. Mol. Biol. 185, 295-309[Medline] [Order article via Infotrieve]
35. Rehrauer, W. M., Bruck, I., Woodgate, R., Goodman, M. F., and Kowalczykowski, S. C. (1998) J. Biol. Chem. 273, 32384-32387[Abstract/Free Full Text]
36. Frank, E. G., Hauser, J., Levine, A. S., and Woodgate, R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8169-8173[Abstract/Free Full Text]
37. Freitag, N., and McEntee, K. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8363-8367[Abstract]
38. Peat, T. S., Frank, E. G., McDonald, J. P., Levine, A. S., Woodgate, R., and Hendrickson, W. A. (1996) Nature 380, 727-730[CrossRef][Medline] [Order article via Infotrieve]
39. Peat, T. S., Frank, E. G., McDonald, J. P., Levine, A. S., Woodgate, R., and Hendrickson, W. A. (1996) Structure 4, 1401-1412[Medline] [Order article via Infotrieve]
40. Roca, A. I., and Cox, M. M. (1997) Prog. Nucleic Acid Res. Mol. Biol. 56, 129-223[Medline] [Order article via Infotrieve]
41. Register, J., III, and Griffith, J. (1985) J. Biol. Chem. 260, 12308-12312[Abstract/Free Full Text]
42. Stasiak, A., Stasiak, A. Z., and Koller, T. (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 745-749[Medline] [Order article via Infotrieve]
43. Griffith, J. D., Harris, L. D., and Register, J., III (1984) Cold Spring Harbor Symp. Quant. Biol. 49, 553-559[Medline] [Order article via Infotrieve]
44. Lohman, T. M., and Ferrari, M. E. (1994) Annu. Rev. Biochem. 63, 527-570[CrossRef][Medline] [Order article via Infotrieve]
45. Muniyappa, K., Williams, K., Chase, J. W., and Radding, C. M. (1990) Nucleic Acids Res. 18, 3967-3973[Abstract]
46. Williams, J. G., Shibata, T., and Radding, C. M. (1981) J. Biol. Chem. 256, 7573-7582[Abstract/Free Full Text]
47. Sommer, S., Bailone, A., and Devoret, R. (1993) Mol. Microbiol. 10, 963-971[Medline] [Order article via Infotrieve]
48. Boudsocq, F., Campbell, M., Devoret, R., and Bailone, A. (1997) J. Mol. Biol. 270, 201-211[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.