A Critical Role of the 3′ Terminus of Nascent DNA Chains in Recognition of Stalled Replication Forks

Abstract

Arrest of replication forks by various internal and external threats evokes a myriad of cellular reactions, collectively known as DNA replication checkpoint responses. In bacteria, PriA is essential for restoration of stalled replication forks and recombinational repair of double-stranded DNA breaks and is a candidate sensor protein that may recognize arrested forks. Here, we report that PriA protein specifically recognizes 3′ termini of arrested nascent DNA chains at model stalled replication forks in vitro. Mutations in the putative “3′ terminus binding pocket” present in the N-terminal segment of PriA result in reduced binding to stalled replication fork structures and loss of its biological functions. The results suggest a mechanism by which stalled replication forks are recognized by a sensor protein for checkpoint responses. Arrest of replication forks by various internal and external threats evokes a myriad of cellular reactions, collectively known as DNA replication checkpoint responses. In bacteria, PriA is essential for restoration of stalled replication forks and recombinational repair of double-stranded DNA breaks and is a candidate sensor protein that may recognize arrested forks. Here, we report that PriA protein specifically recognizes 3′ termini of arrested nascent DNA chains at model stalled replication forks in vitro. Mutations in the putative “3′ terminus binding pocket” present in the N-terminal segment of PriA result in reduced binding to stalled replication fork structures and loss of its biological functions. The results suggest a mechanism by which stalled replication forks are recognized by a sensor protein for checkpoint responses. Progression of replication forks is stalled by a variety of internal and external causes, including DNA damage and depletion of precursors for DNA synthesis. The arrested replication forks elicit checkpoint responses that enable cells to repair damage, restore replication forks, and restart DNA replication (1Cox M.M. Goodman M.F. Kreuzer K.N. Sherratt D.J. Sandler S.J. Marians K.J. Nature. 2000; 404: 37-41Crossref PubMed Scopus (866) Google Scholar, 2Boddy M.N. Russell P. Curr. Biol. 2001; 11: R953-R956Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). A number of eukaryotic gene products have been identified that participate in this process. The initial phase of this response is the recognition of the arrested forks by a sensor protein, which may be recruited to the site of the fork arrest and transduce various signals for further downstream cellular responses. However, the nature of the protein that detects the stalled replication forks and the mechanism by which they are recognized are still unclear. In Escherichia coli, PriA, a DEXH-type DNA helicase originally discovered as a protein essential for replication of a small single-stranded phage DNA, ϕX174, is believed to play a key role in rescuing the stalled replication forks (1Cox M.M. Goodman M.F. Kreuzer K.N. Sherratt D.J. Sandler S.J. Marians K.J. Nature. 2000; 404: 37-41Crossref PubMed Scopus (866) Google Scholar, 3Lee E.H. Masai H. Allen Jr., G.C. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4620-4624Crossref PubMed Scopus (41) Google Scholar, 4Nurse P. DiGate R.J. Zavitz K.H. Marians K.J. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4615-4619Crossref PubMed Scopus (54) Google Scholar, 5Marians K.J. Trends Biochem. Sci. 2000; 25: 185-189Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The priA null cells display numerous phenotypes, including low viability and sensitivity to DNA damaging agents such as UV, γ-ray, and mitomycin C (6Lee E.H. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3029-3032Crossref PubMed Scopus (104) Google Scholar, 7Nurse P. Zavitz K.H. Marians K.J. J. Bacteriol. 1991; 173: 6686-6693Crossref PubMed Google Scholar, 8Masai H. Asai T. Kubota Y. Arai K. Kogoma T. EMBO J. 1994; 13: 5338-5345Crossref PubMed Scopus (93) Google Scholar, 9Kogoma T. Cadwell G.W. Barnard K.G. Asai T. J. Bacteriol. 1996; 178: 1258-1264Crossref PubMed Google Scholar, 10Sandler S.J. Samra H.S. Clark A.J. Genetics. 1996; 143: 5-13Crossref PubMed Google Scholar). These defects may reflect a critical role played by PriA in resumption of DNA replication after arrest of ongoing replication forks (11Sandler S.J. Marians K.J. J. Bacteriol. 2000; 182: 9-13Crossref PubMed Scopus (176) Google Scholar). In E. coli, in fact, arrest of ongoing replication forks induces a recombination-dependent mode of DNA replication, known as inducible or constitutive stable DNA replication (iSDR 1The abbreviations used are: iSDR, inducible stable DNA replication; cSDR, constitutive stable DNA replication; A-fork, arrested fork; HSQC, heteronuclear single quantum coherence.1The abbreviations used are: iSDR, inducible stable DNA replication; cSDR, constitutive stable DNA replication; A-fork, arrested fork; HSQC, heteronuclear single quantum coherence. or cSDR, respectively), which requires PriA protein (8Masai H. Asai T. Kubota Y. Arai K. Kogoma T. EMBO J. 1994; 13: 5338-5345Crossref PubMed Scopus (93) Google Scholar). It was shown that the ϕX174-type primosome could be assembled on a model D-loop structure in vitro (12Liu J. Marians K.J. J. Biol. Chem. 1999; 274: 25033-25041Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 13Liu J. Xu L. Sandler S.J. Marians K.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3552-3555Crossref PubMed Scopus (116) Google Scholar). It has been proposed that PriA may recognize recombination intermediates generated as a result of fork arrest as well as the arrested replication fork per se and facilitate reassembly of replication forks (8Masai H. Asai T. Kubota Y. Arai K. Kogoma T. EMBO J. 1994; 13: 5338-5345Crossref PubMed Scopus (93) Google Scholar, 14Jones J.M. Nakai H. Mol. Microbiol. 2000; 36: 519-527Crossref PubMed Scopus (30) Google Scholar, 15Gregg A.V. McGlynn P. Jaktaji R.P. Lloyd R.G. Mol. Cell. 2002; 9: 241-251Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Consistent with this, PriA recognizes and binds specifically to DNA structures such as those mimicking D-loop (intermediates of homologous recombination reactions) or arrested replication forks (16McGlynn P. Al-Deib A.A. Liu J. Marians K.J. Lloyd R.G. J. Mol. Biol. 1997; 270: 212-221Crossref PubMed Scopus (164) Google Scholar, 17Nurse P. Liu J. Marians K.J. J. Biol. Chem. 1999; 274: 25026-25032Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 18Masai H. Deneke J. Furui Y. Tanaka T. Arai K. Biochimie (Paris). 1999; 81: 847-857Crossref PubMed Scopus (17) Google Scholar). However, it has not been known what structural features of D-loop or arrested DNA replication fork structures are recognized by PriA. In this report, we show that PriA recognizes the 3′-end of DNA and have identified critical residues for this recognition within the N-terminal DNA binding domain. We propose the presence of a 3′ terminus binding pocket on PriA protein, which plays a critical role in detecting stalled replication forks. Purification of Protein Samples—Wild-type PriA-(1-181) and mutant PriA-(1-181) were expressed and purified as previously described (19Tanaka T. Mizukoshi T. Taniyama C. Kohda D. Arai K. Masai H. J. Biol. Chem. 2002; 277: 38062-38071Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). PriA-(1-105) was prepared in the same manner as PriA-(1-181). The N-terminal histidine tag of these proteins was removed by thrombin treatment (1/100, w/w) for BIAcore and NMR analyses. Uniformly 15N-labeled PriA-(1-105) was prepared from E. coli cells carrying an expression plasmid grown in M9 minimal medium containing 1 g/liter of 15NH4Cl. Uniformly 15N,13C,2H-labeled PriA-(1-105) was prepared using M9 minimal medium containing 1 g/liter 15NH4Cl, 2.5 g/liter U-13C glucose in 99% 2H2O. To exchange the amide deuteriums to protons, the purified protein was denatured in 6 m guanidine hydrochloride, followed by dialysis against a buffer solution (25 mm sodium phosphate (pH 5.5), 150 mm KCl, and 10 mm dithiothreitol) for refolding. The protein was repurified by HT-heparin-agarose column (Amersham Biosciences). The level of deuterium incorporation for non-exchangable hydrogens was determined to be about 83% by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. RecG protein was purified from a strain bearing pAF201, as described previously (20Fukuoh A. Iwasaki H. Ishioka K. Shinagawa H. EMBO J. 1997; 16: 203-209Crossref PubMed Scopus (60) Google Scholar). BIAcore Analyses of DNA Binding—The streptavidin (SA) sensor chips with less nonspecific interaction were prepared as follows. A CM5 sensor chip (BIACORE) was activated by N-hydroxysuccinimide/N-ethyl-N′-(3-dimethyl aminopropyl)-carbodiimide hydrochloride (NHS/EDC) solution for 40 min. SA (50 μg/ml in 10 mm sodium acetate (pH 5.3)) was injected onto the activated CM5 surface. The remaining N-hydroxysuccinimide ester groups were inactivated by treatment in 1 m ethanolamine solution for another 40 min, and SA non-specifically adsorbed on the chip was removed by three 1-min washes in 50 mm NaOH and 1 m NaCl, resulting in reduced level of undesired free carboxyl groups. The HBS-EP buffer (10 mm HEPES (pH 7.4), 150 mm NaCl, 3 mm EDTA, and 0.005% (v/v) surfactant P20) was run at 37 °C at a flow rate of 8 μl/min for immobilization. A 17-mer oligonucleotide with/without terminal phosphate modification was immobilized on an SA-coated sensor chip via a biotin moiety attached on either its 5′- or 3′-end. See Fig. 1A for details. The amount of the immobilized oligonucleotide was adjusted to about 150 resonance unit (RU). HBS-EP buffer was used at a flow rate of 30 μl/min at 20 °C for measurement of protein binding. Regeneration of the sensor surface was achieved by three 5-s washes with 10 mm NaOH. The sensorgrams obtained were corrected by subtraction of those recorded by injection of the buffer alone. The dissociation constants were determined by a global fitting method using the BIAevaluation software 3.1 on the basis of the BIAcore experiments using the sensor chips prepared as above which show low nonspecific interactions. NMR Spectroscopy—The PriA-(1-105) was dissolved in 50 mm sodium phosphate buffer (pH 5.5) containing 150 mm KCl, 5 mm dithiothreitol, and 5% 2H2O (v/v). 1H-15N HSQC spectra were measured on a Bruker DMX600 spectrometer at 25 °C. For the assignment of cross-peaks, a series of three-dimensional spectra were recorded using 2H,15N,13C-labeled PriA-(1-105). Gel Shift Assays for DNA Binding of PriA and ATPase Assays—The D-loop and arrested fork-like substrates were prepared by annealing of oligonucleotides using oligonucleotides listed in the table (Table S1) of the Supplementary Materials, followed by isolation from polyacrylamide gel, as described previously (18Masai H. Deneke J. Furui Y. Tanaka T. Arai K. Biochimie (Paris). 1999; 81: 847-857Crossref PubMed Scopus (17) Google Scholar). The gel shift assays were conducted as described previously (19Tanaka T. Mizukoshi T. Taniyama C. Kohda D. Arai K. Masai H. J. Biol. Chem. 2002; 277: 38062-38071Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) except that the concentration of the salt (potassium glutatmate) was reduced to 40 mm in all the experiments other than Fig. 1B. ATPase assays were conducted as described previously (19Tanaka T. Mizukoshi T. Taniyama C. Kohda D. Arai K. Masai H. J. Biol. Chem. 2002; 277: 38062-38071Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), except that D-loop DNA was included as an effector DNA at 4 nm. E. coli Strains—AQ634 (priA +) and AQ8851 (priA1::kan) were previously described (8Masai H. Asai T. Kubota Y. Arai K. Kogoma T. EMBO J. 1994; 13: 5338-5345Crossref PubMed Scopus (93) Google Scholar). E. coli cells were maintained on LB, M9 containing casaamino acids (0.5%; M9-CAA), or M9 containing required amino acids (M9-AA). Transformation of plasmid DNA was conducted by the Inoue method (21Inoue H. Nojima H. Okayama H. Gene (Amst.). 1990; 96: 23-28Crossref PubMed Scopus (1554) Google Scholar). SDR was measured as described before (8Masai H. Asai T. Kubota Y. Arai K. Kogoma T. EMBO J. 1994; 13: 5338-5345Crossref PubMed Scopus (93) Google Scholar, 9Kogoma T. Cadwell G.W. Barnard K.G. Asai T. J. Bacteriol. 1996; 178: 1258-1264Crossref PubMed Google Scholar). We have shown via gel shift assays that the N-terminal 181 residue polypeptide of PriA can bind to a D-loop mimic DNA in vitro (19Tanaka T. Mizukoshi T. Taniyama C. Kohda D. Arai K. Masai H. J. Biol. Chem. 2002; 277: 38062-38071Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). To determine the affinity of PriA and its truncated polypeptides with various forms of DNA, we developed a BIAcore assay system where the biotin-labeled substrate DNA was immobilized on a sensor chip through streptoavidin (Fig. 1A). The PriA N-terminal polypeptide exhibited binding not only to structured DNA mimicking D-loop (data not shown), but also to single-stranded oligonucleotide (17-mer) immobilized to a sensor chip through its 5′-end with K d = 4.9 μm. In contrast, the same oligonucleotide immobilized through its 3′-end did not serve as a ligand. This suggested a possibility that the 3′ terminus may play a crucial role in recognition of DNA by PriA protein. Therefore, we next generated an oligonucleotide whose 3′-end was phosphorylated and immobilized it on a sensor chip through its 5′-end (Fig. 1A). PriA interacted with this 3′-phosphorylated oligonucleotide only very weakly, supporting our hypothesis that a 3′ terminus is a critical determinant for DNA binding of PriA. The above results suggest a possibility that PriA recognizes the ends of non-elongated DNA chains resulting from arrested replication forks or recombination intermediates. To directly test this hypothesis, we conducted gel shift assays with structured DNA mimicking these structures. First, we generated a D-loop-like structure bearing either a 3′-invading or a 5′-invading strand at the fork and examined the binding of PriA by gel shift assays (16McGlynn P. Al-Deib A.A. Liu J. Marians K.J. Lloyd R.G. J. Mol. Biol. 1997; 270: 212-221Crossref PubMed Scopus (164) Google Scholar, 19Tanaka T. Mizukoshi T. Taniyama C. Kohda D. Arai K. Masai H. J. Biol. Chem. 2002; 277: 38062-38071Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). PriA bound to the 3′-invading substrate with much higher affinity (Fig. 1B, upper panel, lanes 1-6 and 13-18). PriA does not form a stable complex with a single-stranded oligonucleotide with a free 3′-end in gel shift assays, and therefore, the presence or absence of a free 3′-end of the invading strand in the 5′-invading D-loop does not affect the gel shift pattern (Fig. 1B, upper panel, lanes 13-18 and 19-24). We then generated a similar D-loop substrate with a 3′-invading strand whose 3′-end was phosphorylated. PriA formed rather smeared complexes on this substrate, and the K d was estimated to be more than of than that for the substrate (Fig. 1B, upper panel, lanes DNA substrates more mimicking arrested fork-like structures were were structures carrying either a nascent strand or a nascent strand or at the fork or PriA generated a complex in gel shift assays with or with affinity d = and Fig. lanes and It bound to with less affinity K d of 8 Fig. lanes the 3′ terminus of the strand was by affinity was reduced to the level similar to that of d = Fig. lanes of of the strand was further substrates were used (Fig. K d = and for and PriA bound to a structure with K d = and of the 3′ terminus at the to reduced the affinity Fig. These results that the 3′ terminus of DNA present at the to is for affinity binding of PriA to fork structures and our hypothesis that PriA protein specifically recognizes the 3′ terminus of the nascent strand at the to be generated from arrest of replication fork RecG has been in the of stalled replication forks in with PriA A.V. McGlynn P. Jaktaji R.P. Lloyd R.G. Mol. Cell. 2002; 9: 241-251Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, P. Lloyd R.G. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar, S. Cell. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). It is also a DEXH-type DNA helicase and is in Lloyd R.G. J. Bacteriol. 1999; PubMed Google Scholar). RecG was shown to bind to structured including the and D-loop-like structures (16McGlynn P. Al-Deib A.A. Liu J. Marians K.J. Lloyd R.G. J. Mol. Biol. 1997; 270: 212-221Crossref PubMed Scopus (164) Google Scholar, Lloyd R.G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). To the recognition of a 3′ terminus in an arrested fork-like structure is to PriA, we have purified RecG and examined its binding to the above RecG protein bound more to the 5′-invading D-loop of the presence or absence of than to the 3′-invading D-loop (Fig. 1B, panel, lanes 1-6 and it bound to the 3′-invading D-loop with a phosphorylated 3′-end as as that with a (Fig. 1B, panel, lanes recognition of a 3′ terminus to be to PriA. This is with the absence of in the N-terminal of PriA and report on measurement of interaction of E. coli PriA protein with single-stranded DNA that this interaction is of DNA from to S. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), which is PriA recognizes an of We have further truncated the N-terminal 181 residue DNA binding segment and by BIAcore measurement that the N-terminal residue segment could bind to the 17-mer oligonucleotide in a manner on the presence of a free 3′ its binding affinity was than PriA-(1-181) d = 20 and not shown), and D-loop binding could not be in gel shift We then binding of PriA-(1-105) to an carrying a free terminus or a phosphorylated 3′ by The DNA, but not the 3′-phosphorylated DNA, in the 1H-15N HSQC of 15N-labeled PriA-(1-105) (Fig. In we have identified and as residues whose the to the exchange of the shift of in various that these residues are a of well residues present very the terminus (Fig. and are well The of the was previously (19Tanaka T. Mizukoshi T. Taniyama C. Kohda D. Arai K. Masai H. J. Biol. Chem. 2002; 277: 38062-38071Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). The of the DNA on the of the suggest the recognition of the of single-stranded DNA by the N-terminal segment of the PriA protein through a binding The three amino acids identified are candidate residues in this putative binding We and or and or and purified the mutant N-terminal was also for it is a residue that we may with through We could not and mutant polypeptides to of NMR spectra that the of the purified mutant proteins was maintained (data not the mutant polypeptides binding and or reduced binding to the 17-mer DNA in the BIAcore (Fig. This result that these residues a of a putative “3′ terminus binding pocket” of PriA protein. We then to PriA carrying in the putative 3′ terminus binding We and purified of and We have shown by gel the purified as well as these mutant proteins as and similar pattern (data not shown), that the structures are not by these bound to D-loop and with affinity than that of the (Fig. We also examined ATPase of the mutant protein. PriA in the presence of its such as or D-loop T. Taniyama C. Arai K. Masai H. PubMed Scopus Google Scholar). Consistent with low affinity of PriA to D-loop with a 3′-phosphorylated 3′-invading PriA rather in the presence of this effector DNA with the In contrast, and low ATPase in the presence of a D-loop structure (Fig. This also the of the 3′ terminus binding pocket for interaction of PriA with its We propose that binding of PriA to arrested forks or structures may be by recognition of the 3′ terminus of the strand or invading strand of an arrested fork or with its putative 3′ terminus binding This which is rather in E. coli, be further by the residues by the that but not is to form a complex with D-loop in gel shift and DNA helicase which with other structures of arrested forks (19Tanaka T. Mizukoshi T. Taniyama C. Kohda D. Arai K. Masai H. J. Biol. Chem. 2002; 277: 38062-38071Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). stable interaction with single-stranded DNA was for PriA protein P. S. S. C. 2002; PubMed Scopus Google Scholar). We expressed the mutant PriA proteins in the strain and examined biological The strain very low and is to DNA damaging agents such as or γ-ray, and of the cells are (6Lee E.H. Kornberg A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3029-3032Crossref PubMed Scopus (104) Google Scholar, 7Nurse P. Zavitz K.H. Marians K.J. J. Bacteriol. 1991; 173: 6686-6693Crossref PubMed Google Scholar, 9Kogoma T. Cadwell G.W. Barnard K.G. Asai T. J. Bacteriol. 1996; 178: 1258-1264Crossref PubMed Google Scholar). This strain is in and (8Masai H. Asai T. Kubota Y. Arai K. Kogoma T. EMBO J. 1994; 13: 5338-5345Crossref PubMed Scopus (93) Google Scholar). The PriA protein expressed from a can these However, and expressed on the same did not restore and SDR (Fig. and Supplementary Fig. which bound to an arrested fork structure with affinity about the could restore of these but to the level of the The 3′ terminus binding of these mutant proteins may with an of DNA with affinity and which may not be in gel shift assays using the proteins carrying other DNA binding and this may result in biological of these to of the priA null mutant was corrected by but only by other (data not to with the pocket may suggest that the forks arrested by could be recognized by these to We also that and could not the of mutant Mol. Microbiol. PubMed Scopus Google and not shown), in with an essential role of the 3′ terminus binding pocket of PriA in of replication forks arrested the of DNA and recognition of the 3′ terminus of an arrested replication fork or that of the invading strand within D-loop by the PriA N-terminal segment is critical for various biological responses by arrest of ongoing replication forks. results suggest that PriA specifically recognizes the replication forks with an arrested the strand a the strand may for a in the absence of strand synthesis. This of strand was previously in plasmid DNA replication E. E. Mol. PubMed Scopus Google Scholar). It may be arrested to on the fork a non-elongated DNA of the nascent double-stranded breaks are generated at the site of arrested forks, of recombination proteins recombination which also an from the invading strand A.V. McGlynn P. Jaktaji R.P. Lloyd R.G. Mol. Cell. 2002; 9: 241-251Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The of PriA to recognize the 3′ terminus of a strand be for and recognition of stalled replication forks, replication forks, or results can the of binding of PriA to the or structures which not a 3′ terminus at the (16McGlynn P. Al-Deib A.A. Liu J. Marians K.J. Lloyd R.G. J. Mol. Biol. 1997; 270: 212-221Crossref PubMed Scopus (164) Google Scholar, 17Nurse P. Liu J. Marians K.J. J. Biol. Chem. 1999; 274: 25026-25032Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). can also the helicase on the strand by the presence of a strand a A.V. McGlynn P. Jaktaji R.P. Lloyd R.G. Mol. Cell. 2002; 9: 241-251Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). the of DNA replication is crucial for of DNA replication of the The recognition of the stalled replication forks is the initial for DNA damage or DNA replication checkpoint responses. It be of eukaryotic sensor proteins recognize the of We for critical of the and We Shinagawa and for of and for E. coli We and for in of PriA protein. We also Taniyama and of for in of mutant PriA and other We and other of our for with