Báo cáo khoa học: Inactivating pentapeptide insertions in the fission yeast replication factor C subunit Rfc2 cluster near the ATP-binding site and arginine finger motif docx

11 468 0
  • Loading ...
    Loading ...
    Loading ...

Tài liệu hạn chế xem trước, để xem đầy đủ mời bạn chọn Tải xuống

Tài liệu liên quan

Thông tin tài liệu

Ngày đăng: 07/03/2014, 02:20

Inactivating pentapeptide insertions in the fissionyeast replication factor C subunit Rfc2 cluster nearthe ATP-binding site and arginine finger motifFiona C. Gray1,2, Kathryn A. Whitehead1,* and Stuart A. MacNeill1,2,1 Wellcome Trust Centre for Cell Biology, University of Edinburgh, UK2 Department of Biology, University of Copenhagen, DenmarkIntroductionThe heteropentameric clamp loader replication factorC (RFC) plays a key role in chromosome replicationin eukaryotic cells. RFC binds to nascent primer–template junctions and catalyses the loading of thering-shaped sliding clamp, proliferating cell nuclearantigen (PCNA), onto DNA [1,2]. The homotrimericPCNA complex encircles the DNA completely, form-ing a sliding clamp that tethers DNA polymerase d tothe DNA, conferring upon it the processivity necessaryto efficiently replicate the genome. PCNA also inter-acts with a large number of additional proteinsimplicated in DNA replication, DNA repair and DNAmodification such as DNA ligase I, the nucleases Fen1and XP-G, uracil-N-glycosylase and cytosine-5-methyl-transferase [3].The five subunits of the RFC complex are related toone another but are not interchangeable [1,2]. Thecomplex comprises a large subunit, Rfc1, and fourKeywordsAAA+protein; clamp loader; DNAreplication; fission yeast; replication factor CCorrespondenceS. MacNeill, Centre for BiomolecularSciences, University of St Andrews, NorthHaugh, St Andrews KY16 9ST, UKFax: +44 01334 462595Tel: +44 01334 467268E-mail: stuart.macneill@st-andrews.ac.ukWebsite: http://biology.st-andrews.ac.uk/macneill/Present addresses*Department of Chemistry and Materials,Manchester Metropolitan University, UKCentre for Biomolecular Sciences,University of St Andrews, UK(Received 20 March 2009, revised 24 June2009, accepted 26 June 2009)doi:10.1111/j.1742-4658.2009.07181.xReplication factor C (RFC) plays a key role in eukaryotic chromosomereplication by acting as a loading factor for the essential sliding clamp andpolymerase processivity factor, proliferating cell nuclear antigen (PCNA).RFC is a pentamer comprising a large subunit, Rfc1, and four smallsubunits, Rfc2–Rfc5. Each RFC subunit is a member of the AAA+familyof ATPase and ATPase-like proteins, and the loading of PCNA onto dou-ble-stranded DNA is an ATP-dependent process. Here, we describe theproperties of a collection of 38 mutant forms of the Rfc2 protein generatedby pentapeptide-scanning mutagenesis of the fission yeast rfc2 gene. Eachinsertion was tested for its ability to support growth in fission yeastrfc2D cells lacking endogenous Rfc2 protein and the location of each inser-tion was mapped onto the 3D structure of budding yeast Rfc2. This analy-sis revealed that the majority of the inactivating mutations mapped in oradjacent to ATP sites C and D in Rfc2 (arginine finger and P-loop, respec-tively) or to the five-stranded b sheet at the heart of the Rfc2 protein. Bycontrast, nonlethal mutations map predominantly to loop regions or to theouter surface of the RFC complex, often in highly conserved regions of theprotein. Possible explanations for the effects of the various insertions arediscussed.AbbreviationsPCNA, proliferating cell nuclear antigen; PSM, pentapeptide-scanning mutagenesis; RFC, replication factor C.FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBS 4803smaller subunits, Rfc2–Rfc5. Genetic analysis in yeasthas shown that each of the five subunits is individuallyessential for chromosome replication [4]. Three RFC-like complexes have also been identified in eukaryoticcells with diverse but poorly understood roles in vari-ous aspects of checkpoint control, cohesion establish-ment and genome stability [5]. In these complexes,Rfc1 is replaced by one of Rad17, Ctf18 or Elg1. Twoadditional subunits are also present in the Ctf18–RFCcomplex.Each of the large and small RFC subunits is a mem-ber of the AAA+family of ATPase and ATPase-likeproteins [6–8], and PCNA loading requires multipleATP hydrolysis events that are discussed furtherbelow. The crystal structure of a variant form of bud-ding yeast RFC bound to PCNA has been solved inthe presence of the nonhydrolysable ATP analogueATP-cS [9]. The crystallized form of the RFC complexlacked N- and C-terminal sequences from Rfc1 (nei-ther of the missing parts of the protein is required forefficient clamp loading in vitro) and carried mutationsin the so-called arginine finger motifs in Rfc2–Rfc5 [9].The five subunits are located in a spiral arrangementin the order Rfc1–Rfc4–Rfc3–Rfc2–Rfc5. At the centreof the spiral, a cavity of sufficient size to accommodatethe primer–template duplex is found. The pitch of thespiral matches that of the DNA, leading to a model inwhich RFC threads onto the 3¢OH group of the pri-mer like a screw-cap, preventing further primer exten-sion until PCNA loading has occurred [9]. In the yeastRFC crystal structure, a gap exists between Rfc1 andRfc5 through which single-stranded DNA has beensuggested to exit.PCNA loading onto DNA by RFC is entirely ATPdependent [1,2]. Biochemical analysis has shown thatATP binding, but not hydrolysis, is required forPCNA binding and opening of the PCNA ring. TheRFC–ATP–open PCNA complex then associates withthe primer–template DNA. This association appears totrigger ordered ATP hydrolysis in the different ATP-binding sites of RFC, closure of the PCNA ring andejection of RFC from DNA, leaving the closed ring onthe DNA.ATP is bound at four sites in RFC (designated ATPsites A–D) located at the subunit interfaces [1,2]. Eachsite is bipartite in nature, comprising elements pro-vided by adjacent subunits. Thus ATP site A is com-posed of the ATP-binding P-loop of Rfc1 (also knownas RFC-A) and an arginine residue located in Rfc4(RFC-B). The side chain of the arginine is referred toas an arginine finger and the finger protrudes into theATP-binding site of the neighbouring subunit. Theexact biochemical roles of the arginine fingers have notbeen precisely defined, but may involve sensing ATPbinding in the P-loop and ⁄ or catalysing subsequentATP hydrolysis. The fingers are not required for ATPbinding [10].In this study, we focus on the Rfc2 protein (alsoknown as RFC-D). Rfc2 binds ATP in site D at theRfc2–Rfc5 (RFC-D–RFC-E) interface and contributesan arginine finger to site C at the Rfc3–Rfc2 (RFC-C–RFC-D) interface. Biochemical analysis of the proper-ties of mutant yeast RFC complexes has shown thatthe Rfc2 arginine finger at site C is required for theRFC–ATP–open PCNA complex to bind DNA, lead-ing to the proposal that the conformational changesrequired for RFC to bind the primer–template DNArequire that the Rfc2 arginine finger responds to thepresence of ATP in site C [10]. ADP cannot substitutefor ATP in these reactions.The Escherichia coli clamp loader, the c-complex,loads the b-sliding clamp onto DNA and is broadlyanalogous to RFC [1,2]. On the basis of analysis ofc-complex subunits [11], three positively charged resi-dues in Rfc2 have been proposed to play a direct rolein DNA binding by RFC by interacting with the phos-phate backbone of duplex DNA in the central cavity[12]. Consistent with this proposal, mutation of thesearginine residues (arginines 101, 107 and 175 in yeastRfc2) abolishes loading of RFC–ATP–open PCNAcomplex onto DNA [12].Once loaded onto DNA, closure of the PCNA ringand release of RFC requires ATP hydrolysis. ATPsites C and D are particularly important for this [10].Site C comprises the arginine finger from Rfc2 and theP-loop of Rfc3 (RFC-C), whereas site D comprises theP-loop of Rfc2 (RFC-D) and the arginine finger fromRfc5. Blocking ATP hydrolysis at sites C and D resultsin a significant inhibition of hydrolysis at sites A andB also, whereas blocking sites A and B has less of aneffect on hydrolysis at sites C and D [10]. Takentogether, these results underline the key role of Rfc2(RFC-D) in RFC function.In this report, we describe the results of an extensivemutagenesis study of the Rfc2 protein of the fissionyeast Schizosaccharomyces pombe. Using pentapeptide-scanning mutagenesis [13,14], a total of 38 mutant rfc2alleles were isolated and tested for their ability tosupport chromosome replication in fission yeast cellscarrying a deletion of the endogenous rfc2+gene [15].The majority of the inactivating mutations map inor around ATP sites C and D (arginine finger andP-loop respectively), or in the five-strand parallel bsheet at the core of Rfc2. By contrast, nonlethalmutations map predominantly to loop regions or tothe outer surface of the RFC complex, often in highlyMutagenesis of the RFC small subunit Rfc2 F. C. Gray et al.4804 FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBSconserved regions of the protein. Possible explana-tions for the effects of the various insertions arediscussed.Results and DiscussionPentapeptide-scanning mutagenesisPentapeptide-scanning mutagenesis (PSM) is a rapidmethod for the random insertion of variable fiveamino acid sequences into a target protein [14]. Herethe system based on transposon Tn4330 was used [13].Tn4430 contains cleavage sites for the restrictionenzyme KpnI only 5 bp from its termini and duplicatesfive nucleotides of target site DNA during transposi-tion. By allowing Tn4430 to insert into a target DNA,then digesting the target with KpnI and re-ligating, thebulk of the transposon is deleted, leaving behind only15 bp of sequence derived from the ends of the trans-poson and the target site duplication. Should Tn4430insertion occur within an ORF, the 15-bp insertion willgenerally result in the encoded protein acquiring a fiveamino acid (pentapeptide) insertion. Insertion can alsoresult in the generation of an inframe stop codon, butowing to the sequence constraints imposed by thesequence of the transposon ends, this is a relativelyrare event [13].In this study, a total of 46 independent PSM inser-tion alleles were isolated by allowing Tn4430 to trans-pose into a plasmid carrying the S. pombe rfc2+gene(see Experimental procedures). Table 1 lists all 46alleles and describes the location and nature of thepentapeptide insertions. DNA sequencing revealed thatthe 46 PSM insertions, despite their independentTable 1. Pentapeptide insertion mutants: location and inserted sequences.Insertion number Domain Location Inserted sequence Identical isolatesLow-levelexpressionHigh-levelexpressionK10 I 6–7 Pro-ArgGlyThrProPro-Arg K34 + +K12 I 8–9 Asn-LysGlyTyrProAsn-Lys + +K17 I 27–28 Pro-ArgGlyThrProPro-Lys – )K32 I 36–37 Gln-GlyTyrProSerGln-Glu + +K33 I 42–43 Val-GlyValProGlnVal-Leu + +K33DK27 I 42–46 Val-GlyValProGlnLys-Thr + +K33DK22 I 42–49 Val-GlyValProLeuLeu-Ser + +K27 I 45–46 Lys-GlyValProGlnLys-Thr K24 + +K22 I 48–49 Leu-GlyValProLeuLeu-Ser + +K23 I 49–50 Ser-LysGlyTyrProSer-Asn ) ++K23DK14 I 49–57 Ser-LysGlyTyrPro -Phe ))K3 I 50–51 Asn-ArgGlyThrProAsn-Asn K4, K6, K7, K30, K31 + +K14 I 57 Phe-Ter K16, K29, K36 ))K18 I 64–65 Gly-ArgGlyThrProGly-Lys ))K19 I 67–68 Ser-ArgGlyThrProSer-Thr ))K9 I 82–83 Met-GlyTyrProLeuMet-Lys K25 + +K11 I 94–95 Glu-GlyValProHisGlu-Arg + +K20 I 99–100 Ile-ArgGlyThrProIle-Ile + +F46 I 124–125 Phe-GlyValProLeuPhe-Lys + +F49 I 124–125 Phe-ArgGlyThrProPhe-Lys + +F45 I 146–147 Thr-ArgGlyThrProThr-Met ))K13 I 149 Ser-Ter ))F37 I 157–158 Cys-LeuGlyTyrProCys-Leu F38 ))F39 I 170–171 Leu-ArgGlyThrProLeu-Ser ))K5 I 171–172 Ser-GlyValProLeuSer-Ser ))F42 I 174–175 Cys-ArgGlyThrProCys-Ser ))K26 II 183–184 Asp-ArgGlyThrProAsp-Asn K28 + +K1 II 195–196 Ala-GlyValProLeuAla-Ala ) ++F41 III 242–243 Val-GlyValProArgVal-Glu ) +K35 III 251–252 Tyr-ArgGlyThrProTyr-Asn + +K15 III 254–255 Ile-ArgGlyThrProIle-Arg + +F44 III 258–259 Leu-GlyValProLeuLeu-Asp + +F47 III 305 Lys-GYPSKVQNIHETF-Ter F48, F50 + +K8 III 331–332 Asp-GlyValProLeuAsp-Leu ) +F. C. Grayet al. Mutagenesis of the RFC small subunit Rfc2FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBS 4805origins, corresponded to only 31 different alleles (inser-tions indicated in bold type in Table 1).In addition to these insertions, generated by straight-forward Tn4430 transposition and excision, sevenmore alleles were constructed by further manipulationof the original set of mutants. These fell into two clas-ses. Three alleles were constructed bearing shortsequence deletions by ligating together theand 3¢portions of different insertion alleles. For example, byligating sequences 5¢ to the KpnI site in rfc2-K33 tosequences 3¢ to the KpnI site of rfc2-K27, a new allele(rfc2-K33D27) was created encoding a protein in whichthose amino acid residues between the K33 and K27insertion sites were deleted (Table 1). Similar alleleswere constructed using rfc2-K33 and rfc2-K22 (rfc2-K33D22) and using rfc2-K23 and rfc2-K14 (rfc2-K23D14). Note that construction of these alleles wasonly possible because the parental Tn4430 insertionswere in the same reading frame. Note also that in thecase of rfc2-K14, the stop codon created by the origi-nal insertion is lost in fusion to rfc2-K23 (Table 1).Also, the unique KpnI site remaining followingTn4430 excision was utilized to insert extra sequencesto expand the pentapeptide insertions by 5, 10 or 20residues. Four such alleles were created in this way:sequences encoding five or ten extra amino acids wereinserted into the KpnI site of rfc2-K26 to create rfc2-K26a and rfc2-K26b, sequences encoding 10 extraamino acids were inserted into rfc2-K1 to create rfc2-K1a and sequences encoding 20 extra amino acids intorfc2-K11 to create rfc2-K11a (see Table 2 for details).Combining these seven new alleles with the original 31pentapeptide insertions gave a total of 38 mutantalleles spread throughout the rfc2 gene (Fig. 1).Expression and functional analysis in fissionyeastIn order to facilitate expression and analysis of themutant proteins in fission yeast, each mutant allelewas cloned 3¢ to the thiamine-repressible nmt1 pro-moter in the expression vector pREP3XH6 [16] andTable 2. Extended insertions: location and inserted sequences.Insertion number Domain Location Inserted sequence Low-level expression High-level expressionK11a I 94–95 Glu-GlyValProProGlyLeuValProProGlyLeuValProThrProGlyValProProGlyLeuValPheHisGlu-Arg) +K26a II 183–184 Asp-ArgGlyThrProGlyValGlyThrProAsp-Asn++K26b II 183–184 Asp-ArgGlyThrProGlyGlyValGlyProGlyValGlyThrProAsp-Asn++K1a II 195–196 Ala-GlyValProProValGlyLeuGlyProGlyLeuValProLeuAla-Ala++Fig. 1. Location of pentapeptide insertions in Rfc2 protein. Schematic representation of the fission yeast Rfc2 protein showing the locationof the pentapeptide insertion mutants generated in this study. Light grey box: domain I (amino acids 1–181). White box: domain II (aminoacids 182–246). Dark grey box: domain III (amino acids 247–340). Open circles: functional proteins. Grey filled circles: partly functionalproteins. Black circles: non-functional proteins. See text, Table 1 and Fig. 2 for further details.Mutagenesis of the RFC small subunit Rfc2 F. C. Gray et al.4806 FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBSeach plasmid transformed individually into an S. pom-be rfc2+⁄ rfc2::ura4+diploid strain [15]. Individualtransformant colonies were then induced to sporulateand the properties of the meiotic products examinedfollowing growth on minimal medium in the presenceor absence of thiamine, i.e. with the nmt1 promotereither repressed or derepressed (see Experimental pro-cedures for further details). Figure 1 summarizes theproperties of the Rfc2 mutants determined from thisanalysis.When overexpressed (in cells grown in the absenceof thiamine to fully induce the nmt1 promoter), 27 ofthe 38 mutant proteins were able to substitute forloss of wild-type Rfc2 function (20 insertions shownas unfilled or shaded circles in Fig. 1 plus three dele-tion alleles shown in Table 1 and four extended inser-tion alleles shown in Table 2). This decreased to 24when thiamine was present in the growth medium,because three of the mutant alleles could only rescuewhen the Rfc2 protein was overproduced (shaded cir-cles in Fig. 1, plus the K11a extended insertion, seeTable 2). Note that viable mutant strains were alsoexamined for increased sensitivity to the DNA repli-cation inhibitor hydroxyurea, which might indicate aspecific defect in replication checkpoint function.However, none of the strains displayed this property(data not shown). In the following discussion, themutations are considered in three groups dependenton their location in the 3D structure of the Rfc2protein.Mapping the insertions onto the structure ofRfc2: insertions into the N-terminal AAA+domainThe 3D structure of the Rfc2 protein comprises threeseparate domains. Domains I and II together form theAAA+ATPase module, whereas domain III formspart of the RFC circular collar and is unique toclamps loaders [9]. Based on alignment with the bud-ding yeast Rfc2 protein, the N-terminal AAA+domain of fission yeast Rfc2 encompasses amino acidresidues 1–181 (Fig. 1). In the Rfc2 crystal structure(PDB entry 1SXJ) this domain comprises 11 a-helicalsegments and 5 b strands [9]. Here the helices are des-ignated a1–a3, a3¢ and a4–a10 and the strands b1–b5(Fig. 2A). Note that in PDB entry 1SXJ, the a3–a3¢segment is designated as a single a helix (helix 69)despite there being clear structural discontinuity at res-idues 84–85. For this reason, residues 70–89 aredenoted here as two separate a helices, a3 and a3 ¢(Fig. 2A).Twenty-three insertions were located in this domainof the protein, the most N-terminal (K10) beinglocated between residues 6 and 7, and the most C-ter-minal (F42) between residues 174 and 175. The aver-age distance between the insertions is eight residues,but some clustering was observed (Figs 1 and 2A) andthe longest stretch without an insertion is 25 residues(amino acids 100–124 inclusive).All 10 inactivating mutations identified in this studywere located in the N-terminal AAA+domain(Fig. 1). Three of these (insertions K17, K18, K19)were located in or around the P-loop that forms partof RFC ATP-binding site D (Fig. 3, left-hand panel).Insertions K18 and K19 are located in helix a3 closeto the a-phosphate of ATP, whereas insertion K17maps to the unstructured loop region that lies betweena1 and a2, close to the adenosine of bound ATP(Figs 2A and 3). It is not unreasonable to postulatethat these three mutations exert their effects by dis-rupting ATP binding in site D, thereby rendering theRFC complex inactive.Four of the remaining inactivating mutations (K5,F39, F42, F45) cluster close to the Rfc2 arginine finger(Fig. 3, right-hand panel) that forms part of ATP-binding site C at the interface of Rfc2 and Rfc3 (alsoknown as RFC-C). As noted above, the arginine fingerat this site is crucial for the association of the RFC–ATP–open PCNA complex with the primed template,and subsequent ATP hydrolysis, but not for ATPbinding [10]. Insertions K5 and F42 flank the highlyconserved SRC motif containing the arginine (RFCbox VII), whereas insertion F39 is located one aminoacid N-terminal to SRC. Both F39 and K5 insertionsdisrupt the a10 helix, whereas insertion F45 disruptsa8. These insertions are likely to affect the positioningof the arginine finger in site D, thereby blocking DNAbinding by RFC–PCNA.Of the remaining three inactivating mutations, one(insertion F37) maps to b-strand b4 in the five-strandparallel b sheet that comprises the core of domain I(Figs 2A and 3A). The b4 strand is the central strandin the sheet [9]; it is likely that disruption of this strandby pentapeptide insertion will affect the entire sheet(Fig. 3B). The final two inactivating mutations indomain I (K13, K14) cause premature termination ofthe Rfc2 polypeptide chain.The remaining 13 insertions in domain I failed to dis-rupt Rfc2 function. Given that all the pentapeptideinsertion sequences include a proline residue that mightbe expected to have a significant effect on the secondarystructure in the vicinity of the insertion, it is perhaps sur-prising that only 8 of the 29 pentapeptide insertionsinvestigated in this study (< 30% of the total) abolishedRfc2 function altogether (black circles in Fig. 1; thesefigures exclude the two insertions that resulted in prema-F. C. Gray et al. Mutagenesis of the RFC small subunit Rfc2FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBS 4807AFig. 2. Location of insertion sites in conserved regions. Location of insertions in N-terminal AAA+domain (domain I, shown in A), centraldomain (domain II, B) and C-terminal collar domain (domain III, C) of fission yeast Rfc2. The aligned sequences of 10 Rfc2 proteins fromdiverse eukaryotic species are shown, corresponding to S. pombe Rfc2 residues 1–181 (domain I), 182–241 (domain II) and 242–340 (domainIII). Insertions resulting in premature termination of translation are underlined. Abbreviations and RefSeq accession numbers: Sp (S. pombe,NP_594540), Sc (Saccharomyces cerevisiae, NP_012602), Hs (Homo sapiens, NP_852136), Dm (Drosophila melanogaster , NP_573245), Gg(Gallus gallus, NP_001006550), Xl (Xenopus laevis, NP_001082757), Dr (Danio rerio, NP_999902), At (Arabadopsis thaliana, NP_564148), Dd(Dictyostelium discoideum, XP_637900) and Ez (Encephalitozoon cuniculi, XP_955685). Positions conserved in at least eight of the ten pro-teins are shown boxed. Secondary structure elements in S. cerevisiae Rfc2 are shown above (based on PDB entry 1SXJ, see Fig. 3). Thealignments were generated usingCLUSTAL X [22,23].Mutagenesis of the RFC small subunit Rfc2 F. C. Gray et al.4808 FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBSture polypeptide chain termination). Related studies ofother proteins [13,17–19] have revealed similar ratios offunctional to nonfunctional mutants, however, so thisphenomenon is not specific to RFC.Four of the nonlethal insertions in domain I mappedwithin a helices (Fig. 2A): two in the a2 helix (inser-tions K33 and K27), one at the extreme C-terminalend of a3¢ (K9) and one in the middle of a4, close tothe border of the a4 and a5 helices on the inner faceof the RFC complex (K20). Two of the three con-served arginine residues implicated in DNA binding byRfc2 (R101 and R107 in the budding yeast Rfc2 pro-tein, R95 and R101 in the fission yeast protein) arelocated in a4 [12]. If the K20 insertion disrupts eitherof these interactions, it does so without markedly dis-rupting RFC function. Simultaneous mutation of thethree conserved arginine residues to alanines in thebudding yeast Rfc2, Rfc3 and Rfc4 proteins (such thatthe resulting RFC complex carried nine arginine-to-alanine substitutions) abolished DNA binding, but therelative contributions of the individual subunits or theindividual arginines in each subunit were not tested[12].Our results may imply that the a4 arginines R95and R101 are not absolutely required for in vivo DNAbinding by Rfc2 or indeed that DNA binding by Rfc2is nonessential for RFC complex function. Furtherbiochemical and genetic analysis is required to resolvethis issue. However, as a starting point, we used site-directed mutagenesis to construct seven new single,double and triple arginine-to-alanine mutations in fis-sion yeast Rfc2 (designated Rfc2–S1 to Rfc2–S7), atR95 and R101 in the a4 helix and at R165, the thirdresidue previously implicated in DNA binding [12].Each mutant allele (Table 3) was expressed from thenmt1 promoter in haploid rfc2D cells exactly asdescribed above for the pentapeptide insertionmutants. The results of this are summarized inTable 3. All seven mutant proteins, including Rfc2–S7with triple K95A, K101A and K165A substitutions,were able to rescue for loss of Rfc2 function whenexpressed at low level (nmt1 promoter repressed by theBCFig. 2. Continued.F. C. Gray et al. Mutagenesis of the RFC small subunit Rfc2FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBS 4809presence of thiamine in the growth medium, seeabove). Clearly, if DNA binding by Rfc2 does play anessential role in vivo, then the three arginines are notrequired for these interactions to occur. Interestingly,when plated on plates containing 10 mm hydroxyurea,cells expressing Rfc2–S7 were somewhat less elongatedand more heterogeneous in appearance than cellsexpressing wild-type Rfc2 or any of the single or dou-ble arginine-to-alanine mutants Rfc2–S1 to Rfc2–S6,suggesting that Rfc2 function may be mildly impairedin these cells.The remaining nine insertions are located in loopregions where it is reasonable to expect that theinserted sequences would be tolerated without disrupt-ing RFC function (Fig. 2A): insertions are found atthe N-terminus of the protein (K10, K12), between a1and a2 (K32), between a2 and b1 (K22, K23 and K3),between b2 and a4 (K11) and at the N-terminal end ofb3 (F46, F49).Two domain I deletion constructs (K33DK27 andK33D22) were also viable, despite the latter appearingto delete almost all of the a2 helix (Table 1). This helixcontains a pair of very well-conserved basic aminoacids (lysines 44 and 45 in fission yeast Rfc2) and theK33D22 deletion removes these altogether, replacingthe sequence LKKT with GVP (see Fig. 2A andTable 1). The function of a2 helix and the conservedbasic residues is unclear: in the budding yeast RFCcrystal structure, the side chains of the arginines pro-trude from the surface of the RFC complex (Fig.3A),perhaps suggesting an involvement in mediatingprotein–protein or protein–DNA interactions on thesurface of the complex [9]. The a2 helix is not close tothe proposed exit path for single-stranded DNA, how-ever, suggesting that the latter possibility is unlikely.The third deletion mutation tested here (K23DK14)deletes much of the a2–b1 loop region and the b1strand and was not viable. Presumably deleting b1 dis-rupts the structure of the b sheet at the core of domainI (Fig. 3). As noted above, insertion F37 in the b4Table 3. Arginine-to-alanine DNA-binding mutants.Allele Domain Amino acid changesLow-levelexpressionHigh-levelexpressionS1 I R95A + +S2 I R101A + +S3 I R165A + +S4 I R95A-R101A + +S5 I R95A-R165A + +S6 I R101A-R165A + +S7 I R95A-R101A-R165A + +A B Fig. 3. Mapping of insertion sites ontobudding yeast Rfc2 protein structure. (A) 3Dstructure of the budding yeast Rfc2 proteinbound to ATP-cS [9]. The locations of theeight inactivating insertion mutations areindicated by the blue circles. The locationsof the three arginines implicated in DNAbinding are shown as R95, R101 and R165(fission yeast numbering). The coordinatesof Rfc2 were extracted from PDB file 1SXJand the structure drawn usingMACPYMOL0.99 (DeLano Scientific, Palo Alto, CA,USA). ATP-cS is shown in blue and thelocations of the three domains of theprotein indicated. The images of the left andright are rotated by  180° relative to oneanother. (B) Topology diagram of Rfc2 bsheet. For simplicity, a helices are notshown.Mutagenesis of the RFC small subunit Rfc2 F. C. Gray et al.4810 FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBSstrand also eliminates Rfc2 function (Table 1). Noother insertions in the b sheet were identified in thisstudy.Mapping the insertions onto the structure ofRfc2: insertions into the central AAA+domainThe a-helical central domain (domain II) of fissionyeast Rfc2 encompasses amino acid residues 182–246(Figs 1 and 2B). In the yeast RFC structure [9], thiscomprises five a helices (here designated a11–a15).Only three of the insertions were located in thisdomain (Fig. 2B): two of these insertions map withina11 (K26, K1) and the third in a15 (F41). None ofthe mutants abolishes RFC function but rescue ofrfc2D cells by Rfc2–F41 was seen only when the cor-responding mutant gene was overexpressed from thenmt1 promoter, suggesting that the Rfc2–F41 proteinis functionally impaired (Fig. 1). Helix a11 lies onthe outer surface of the RFC complex. Expandingthe K1 and K26 insertions by addition of sequencesencoding a additional 5 or 10 amino acids (seeTable 2 for details) did not disrupt Rfc2 functioneither.Mapping the insertions onto the structure ofRfc2: insertions into the C-terminal collar domainThe C-terminal domain (domain III) of Rfc2 encom-passes amino acid residues 247–340 (Fig. 2C) and com-prises six a helices (a16–a20). These domains form aright-handed spiral collar structure from which theAAA+domains I and II appear to hang [9]. Fiveinsertions map within domain III (Fig. 2C). InsertionsK35, K15 and F44 map within a16, which is locatedon the outer surface of the RFC complex (Fig. 3A),whereas insertion K8 maps centrally within a20located at the Rfc2–Rfc5 interface. None of thesemutations abolishes Rfc2 function but, as with Rfc2–F41 described above, Rfc2–K8 was required to beoverexpressed in order to rescue rfc2D cells, implyingthat the K8 insertion causes a degree of functionalimpairment (Fig. 1).The insertion in F47 is located in a19 (Fig. 2C), butthis mutation is unusual in that Tn4430 transpositionand subsequent restriction enzyme cleavage andre-ligation left behind a 16 bp, rather than a 15 bp,insertion in the DNA sequence. This produces aframe-shift mutation that causes termination of Rfc2following the addition of 13 random amino acids afterlysine 305 (shown in single-letter code in Table 1).Despite this, however, the Rfc2–F47 protein is func-tional, even when expressed at normal levels (Fig. 1and Table 1). It can be concluded from this that a19and a20 are not required for Rfc2 function, regardlessof the negative effect on the Rfc2 protein observedwith insertion K8 in a20 described above. One possibleexplanation for the apparent discrepancy is that theK8 pentapeptide insertion may cause structural disrup-tion that is incompatible with collar formation andRFC function (consistent with the location of the a20helix at the Rfc2–Rfc5 interface), whereas the trun-cated Rfc2–F47 protein forms RFC complexes nor-mally. Further analysis of this point will requiredetailed biochemical analysis of the complex formingproperties of the mutant proteins.ConclusionsThis study confirms the importance of ATP site C(which involves the Rfc2 arginine finger) and ATP siteD (involving the Rfc2 P-loop) for RFC functionin vivo, and demonstrates that three arginine residues(R95, R101 and R165) previously implicated in DNAbinding by Rfc2 are nonessential in vivo. In addition,several highly conserved regions of the Rfc2 proteinthat are surprisingly tolerant of pentapeptide insertionshave been identified. Future work will focus on investi-gating in greater depth the roles these conservedregions play in RFC function.Experimental proceduresBacterial and yeast strains and mediaE.coli DH5a (Stratagene, La Jolla, CA, USA) was used forroutine cloning steps and FH1046 and DS941 for pentapep-tide mutagenesis [13]. E. coli was cultured on LB medium.S. pombe rfc2+⁄ rfc2::ura4+leu1-32 ⁄ leu1-32 ura4-D18 ⁄ ura4-D18 ade6-M210 ⁄ ade6-M216 h)⁄ h+[15] was used for func-tional testing of rfc2 mutations. S. pombe was cultured onYE, EMM or ME media [20] as required, and transformedby electroporation [21].Pentapeptide mutagenesisTo mutagenise rfc2+using the pentapeptide insertionmethod, an rfc2+cDNA was first amplified by PCR from anS. pombe cDNA library using oligonucleotides SPRFC2–5BAM (oligo sequence with BamHI site in lower case andrfc2+start codon underlined: 5¢-TTGGTTGGggatccAAATGTCTTTCTTTGCTCCA-3¢) and SPRFC2–3BAM(oligo sequence with BamHI site in lower case and rfc2+stopcodon underlined: 5¢-TTGGTTGGggatccTTTTCAATGTATAGACTAGC-3¢), restricted with BamHI and cloned intoplasmid pBR322 to generate pBR322–Rfc2. The sequenceF. C. Gray et al. Mutagenesis of the RFC small subunit Rfc2FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBS 4811of the rfc2+insert was confirmed by sequencing. PSM wasperformed using the Tn4430 system [13]. pBR322–Rfc2 wastransformed into E. coli FH1046 and individual transfor-mant colonies were mated with E. coli DS941 after which themating mixes were plated on medium to select for clonescontaining pBR322-Rfc2 carrying the Tn4430 transposon.Isolates containing a Tn4430 insertion within the rfc2 regionwere then identified by colony PCR and plasmid DNAprepared. This was restricted by KpnI and self-ligated toremove the bulk of the transposon, before being trans-formed into E. coli DH5a. Thirty-four individual trans-poson-free clones were isolated in this manner and theposition of the 15-bp insertion determined by KpnI restric-tion site mapping and DNA sequencing. Twelve dupli-cated mutants were discarded at this point, leaving acollection of 22 different pentapeptide insertion alleles(rfc2-K1 to rfc2-K36).A second mutagenesis was carried out using pBR322–Rfc2–SB as the target plasmid. This plasmid was created bycloning the SalI–BamHI fragment carrying the 600 bp3¢ region of the rfc2+ORF from pBR322-Rfc2 intopBR322. pBR322–Rfc2–SB was then subjected to pentapep-tide mutagenesis as above. Twelve independent transposon-containing isolated were identified with insertions in theSalI–BamHI region; sequence analysis showed that theserepresented only nine different insertions. These alleles weredesignated rfc2-F37 to rfc2-F49.Generating deletion and insertion allelesDeletions alleles were constructed by digesting pREP3XH6–Rfc2–K plasmids with KpnI (a second KpnI site is located inthe LEU2 gene) and ligating together complementary pieces.Three alleles were constructed in this way: rfc2-K33 ⁄ K27,rfc2-K33 ⁄ K22 and rfc2-K23 ⁄ K14. Four insertion alleles wereconstructed by digesting the relevant pREP3XH6–Rfc2–Kplasmids with KpnI and ligating in oligonucleotide duplexesconstructed by annealing together the following complemen-tary pairs of oligonucleotides, either SPRFC2-F1 (5¢-CCCCGGGGTTGGTAC-3¢) and SPRFC2-F2 (5¢-CAACCCCGGGGGATG-3¢) to produce a five amino acid extension(insertion K26a) or SPRFC2-F3 (5¢-CCCCGGTGGGGTTGGGCCCGGGGTTGGTAC-3¢) and SPRFC2-F4 (5¢-CAACCCCGGGCCCAACCCCACCGGGGGTAC-3¢) to gen-erate a 10 amino acid extension (insertions K1a and K26b).Insertion K11a resulted from the fortuitous ligation of fourcopies of the F1 ⁄ F2 duplex.Site-directed mutagenesisSite-directed mutagenesis was performed using the PCRoverlap extension mutagenesis with Pfu DNA polymerase(Promega, Madison, WI, USA) and plasmid pREP3XH6–Rfc2 as template. Oligonucleotide sequences are availablefrom the corresponding author on request. Following thesecond round of PCR, the product was digested withBamHI, re-cloned into pREX3XH6 and sequenced to con-firm the absence of PCR errors. The resulting plasmidswere then transformed into yeast as described below.Expression in fission yeastTo express the mutant rfc2-K alleles in S. pombe, eachwas transferred, as a BamHI fragment, into plasmidpREP3XH6 [16]. The resulting pREP3XH6–Rfc2 plasmidsexpress Rfc2 with a 13 amino acid N-terminal extensionthat includes a hexahistidine tag (sequence of extension:MRGSHH HHHHGIQ). For the rfc2-F alleles, a SalI–EcoRV fragment from pBR322–Rfc2–SB (the EcoRV site islocated in the vector sequence,  200 bp from the BamHIsite) was transferred into plasmid pREP3XH6–Rfc2 thathad been cut with SalI and SmaI. The resulting plasmidswere then transformed into S. pombe rfc2+⁄ rfc2::ura4+leu1-32 ⁄ leu1-32 ura4-D18 ⁄ ura4-D18 ade6-M210 ⁄ ade6-M216h)⁄ h+[15] by electroporation [21] and transformantsobtained on EMM medium. Individual colonies were thenpatched overnight at 32 °C on ME medium to induce spor-ulation, before being treated overnight with helicase tobreak down the asci walls and eliminate vegetative cells.Spores were then washed with water before being platedon EMM plates supplemented with adenine (EMM + A),uracil and adenine (EMM + AU), adenine and thiamine(EMM + AT) and adenine, uracil and 5 lm thiamine(EMM + AUT) at 23, 32 and 36.5 °C. Leucine wasomitted from all plates to facilitate selection of pREP3Xplasmids which carry the LEU2 selectable marker. Adenineis required to permit the growth of haploid cells either theade6-M210 or ade6-M216 alleles. The addition of uracilpermits growth of rfc2+haploids; in the absence of uracil,only rfc2::ura4+haploids expressing functional Rfc2proteins can grow. The presence of 5 lm thiamine repressesthe nmt1 promoter in pREP3X, reducing rfc2 expression bya factor of 80–100 compared with cells grown on EMMwithout thiamine.AcknowledgementsWe would like to thank Dr Finbarr Hayes (Universityof Manchester, UK) for supplying the strains necessaryfor PSM. This research was funded by a WellcomeTrust Senior Fellowship in Basic Biomedical Research.KAW was the recipient of a BBSRC-funded postgrad-uate studentship.References1 Indiani C & O’Donnell M (2006) The replicationclamp-loading machine at work in the three domainsof life. Nat Rev Mol Cell Biol 7, 751–761.Mutagenesis of the RFC small subunit Rfc2 F. C. Gray et al.4812 FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBS[...]... for replication factor C in DNA replication checkpoint function in fission yeast Nucleic Acids Res 27, 462–469 Gray FC & MacNeill SA (2000) The Schizosaccharomyces pombe rfc3+ gene encodes a homologue of the human hRFC36 and Saccharomyces cerevisiae Rfc3 subunits of replication factor C Curr Genet 37, 159–167 Hayes F, Hallet B & Cao Y (1997) Insertion mutagenesis as a tool in the modification of protein...F C Gray et al 2 Johnson A & O’Donnell M (2005) Cellular DNA replicases: components and dynamics at the replication fork Annu Rev Biochem 74, 283–315 3 Moldovan GL, Pfander B & Jentsch S (2007) PCNA, the maestro of the replication fork Cell 129, 665–679 4 MacNeill SA & Burgers PMJ (2000) Chromosomal DNA replication in yeast: enzymes and mechanisms In The Yeast Nucleus (Fantes P & Beggs... Martin J (2002) AAA proteins Curr Opin Struct Biol 12, 746–753 9 Bowman GD, O’Donnell M & Kuriyan J (2004) Structural analysis of a eukaryotic sliding DNA clamp-clamp loader complex Nature 429, 724–730 10 Johnson A, Yao NY, Bowman GD, Kuriyan J & O’Donnell M (2006) The replication factor C clamp loader requires arginine finger sensors to drive DNA binding and proliferating cell nuclear antigen loading... Hayes F (1997) Pentapeptide scanning mutagenesis: random insertion of a variable Mutagenesis of the RFC small subunit Rfc2 14 15 16 17 18 19 20 21 22 23 five amino acid cassette in a target protein Nucleic Acids Res 25, 1866–1867 Hayes F & Hallet B (2000) Pentapeptide scanning mutagenesis: encouraging old proteins to execute unusual tricks Trends Microbiol 8, 571–577 Reynolds N, Fantes PA & MacNeill SA (1999)... Insertion mutagenesis as a tool in the modification of protein function Extended substrate specificity conferred by pentapeptide insertions in the omega-loop of TEM-1 beta-lactamase J Biol Chem 272, 28833–28836 Cao Y, Hallet B, Sherratt DJ & Hayes F (1997) Structure–function correlations in the XerD site- speci c recombinase revealed by pentapeptide scanning mutagenesis J Mol Biol 274, 39–53 Zhang Y, Altshuller... loading J Biol Chem 281, 35531–35543 11 Goedken ER, Kazmirski SL, Bowman GD, O’Donnell M & Kuriyan J (2005) Mapping the interaction of DNA with the Escherichia coli DNA polymerase clamp loader complex Nat Struct Mol Biol 12, 183–190 12 Yao NY, Johnson A, Bowman GD, Kuriyan J & O’Donnell M (2006) Mechanism of proliferating cell nuclear antigen clamp opening by replication factor C J Biol Chem 281, 17528–17539... receptor regulation by a phospholipase D1 mutant unresponsive to protein kinase C EMBO J 18, 6339–6348 Moreno S, Klar A & Nurse P (1991) Molecular genetic analysis of fission yeast Schizosaccharomyces pombe Methods Enzymol 194, 795–823 Prentice HL (1992) High-efficiency transformation of Schizosaccharomyces pombe by electroporation Nucleic Acids Res 20, 621 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin... (2004) The PCNA-RFC families of DNA clamps and clamp loaders Prog Nucleic Acid Res Mol Biol 78, 227–260 6 Neuwald AF, Aravind L, Spouge JL & Koonin EV (1999) AAA(+): a class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes Genome Res 9, 27–43 7 Ogura T & Wilkinson AJ (2001) AAA+ superfamily ATPases: common structure – diverse function Genes Cells... Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools Nucleic Acids Res 25, 4876–4882 Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R et al (2007) Clustal W and Clustal X version 2.0 Bioinformatics 23, 2947–2948 FEBS Journal 276 (2009) 4803–4813 ª 2009 The. .. McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R et al (2007) Clustal W and Clustal X version 2.0 Bioinformatics 23, 2947–2948 FEBS Journal 276 (2009) 4803–4813 ª 2009 The Authors Journal compilation ª 2009 FEBS 4813 . Inactivating pentapeptide insertions in the fission yeast replication factor C subunit Rfc2 cluster near the ATP-binding site and arginine finger motif Fiona. produce a five amino acid extension(insertion K26a) or SPRFC2-F3 (5¢-CCCCGGTGGGGTTGGGCCCGGGGTTGGTAC-3¢) and SPRFC2-F4 (5¢-CAACCCCGGGCCCAACCCCACCGGGGGTAC-3¢)
- Xem thêm -

Xem thêm: Báo cáo khoa học: Inactivating pentapeptide insertions in the fission yeast replication factor C subunit Rfc2 cluster near the ATP-binding site and arginine finger motif docx, Báo cáo khoa học: Inactivating pentapeptide insertions in the fission yeast replication factor C subunit Rfc2 cluster near the ATP-binding site and arginine finger motif docx, Báo cáo khoa học: Inactivating pentapeptide insertions in the fission yeast replication factor C subunit Rfc2 cluster near the ATP-binding site and arginine finger motif docx