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A Universal Protein–Protein Interaction Motif in the Eubacterial DNA Replication and Repair Systems

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A Universal Protein–Protein Interaction Motif in the Eubacterial DNA Replication and Repair Systems A universal protein–protein interaction motif in the eubacterial DNA replication and repair systems Brian P. Dalrymple*†, Kritaya Kongsuwan*†, Gene Wijffels*†, Nicholas E. Dixon‡, and Philip A. Jennings†§ †Commonwealth Scientific and Industrial Research Organisation Livestock Industries, 120 Meiers Road, Indooroopilly QLD 4068, Australia; and ‡Research School of Chemistry, Australian National University, Canberra ACT 0200, Australia Communicated by W. James Peacock, Commonwealth Scientific and Industrial Research Organisation, Canberra, Australia, July 24, 2001 (received for review March 9, 2001) The interaction between DNA polymerases and sliding clamp then examined by yeast two-hybrid and peptide-binding exper- proteins confers processivity in DNA synthesis. This interaction is iments with native and modified sequences. critical for most DNA replication machines from viruses and pro- karyotes to higher eukaryotes. The clamp proteins also participate Materials and Methods in a variety of dynamic and competing protein–protein interac- Sources of Amino Acid Sequences. Amino acid sequences and tions. However, clamp-protein binding sequences have not so far alignments were derived from: PSI-BLAST of the protein database been identified in the eubacteria. Here we show from three lines at the National Center for Biotechnology Information (NCBI), of evidence, bioinformatics, yeast two-hybrid analysis, and inhi- and BLAST of preliminary sequence data from NCBI at http:͞͞ bition of protein–protein interaction by modified peptides, that www.ncbi.nlm.nih.gov͞Microb_blast͞unfinishedgenome.html, variants of a pentapeptide motif (consensus QL[SD]LF) are suffi- Institute for Genomic Research at http:͞͞www.tigr.org, Depart- cient to enable interaction of a number of proteins with an ment of Energy Joint Genome Institute at http:͞͞spider.jgi- archetypal eubacterial sliding clamp (the ␤ subunit of Escherichia psf.org͞JGI_microbial͞html͞, Sanger Center at http:͞͞ coli DNA polymerase III holoenzyme). Representatives of this motif www.sanger.ac.uk͞DataSearch͞omniblast.shtml, and ERGO at are present in most sequenced members of the eubacterial DnaE, http:͞͞wit.integratedgenomics.com͞IGwit͞. Alignments of PolC, PolB, DinB, and UmuC families of DNA polymerases and the available sequences of all members of eubacterial protein fam- MutS1 mismatch repair protein family. The component tripeptide ilies known to bind to ␤ were compiled with manual editing in MICROBIOLOGY DLF inhibits the binding of the ␣ (DnaE) subunit of E. coli DNA regions of variable length and sequence. After the identification polymerase III to ␤ at ␮M concentration, identifying key residues. of the proposed ␤-binding motif, alignments of the sequences of Comparison of the eubacterial, eukaryotic, and archaeal sliding members of eubacterial families homologous to eukaryotic clamp binding motifs suggests that the basic interactions have proteins known to bind to proliferating cell nuclear antigen been conserved across the evolutionary landscape. (PCNA) were compiled from GenBank as described above. Regions containing the proposed ␤-binding peptide motifs were ␤ he replication of DNA in eubacteria involves many proteins aligned to maximize matches of a putative -binding site to the Torganized into a complex multifunctional machine termed evolving consensus sequence. the replisome. A central enzyme is the multisubunit DNA polymerase III holoenzyme. In Escherichia coli, and probably in Strains. E. coli XL-1Blue was the host for all plasmid construc- most other eubacteria, the DnaE ortholog (␣ subunit) is in the tions and source of chromosomal DNA. The pLexA, pB42AD, core of the replicative polymerase, whereas in many Gram- p8op-lacZ vectors, and yeast EGY48 cells were from the Match- positive organisms a related enzyme, PolC, is proposed to have maker two-hybrid system (CLONTECH). this function (1). The processivity of the polymerase is conferred Yeast Two-Hybrid Assays. E. coli ␤ dnaN by the direct interaction of the ␤ subunit (clamp protein) of DNA The coding region of ( ) was amplified by PCR (Pfu polymerase) from chromosomal polymerase III (2, 3), with the DnaE (and presumably PolC) DNA and inserted at the EcoRI site of pB42AD to create a subunits. ␤ is loaded onto DNA by a clamp loader comprised of translational fusion. To construct various deletions of the E. coli single ␦ and ␦Ј subunits and four ␶͞␥ subunits (1). The ␤ dimer dnaE, the appropriate portion of dnaE was amplified by PCR thence encircles the DNA without actually binding to it. In and inserted between the EcoRI and XhoI sites of pLexA for an addition to DnaE, three other E. coli DNA polymerases appear in-frame fusion. For site- directed mutagenesis, the dnaE frag- to interact with ␤. PolB (DNA polymerase II) is involved in DNA ment was cloned into pQE11 (Qiagen, Chatsworth, CA), and repair (4) and the addition of ␤ and the clamp loader increases mutations were introduced by using the QuikChange system its processivity in vitro (5, 6). Similarly, ␤ and the clamp loader (Stratagene). The PCR-generated fragments containing the together increase both the processivity (7) and efficiency (8) of mutation then were subcloned into pLexA. To express peptides DNA synthesis by DNA polymerase IV (DinB). ␤ also appears containing the putative ␤-binding regions, appropriate regions to play a similar role in the activity of DNA polymerase V (8) of E. coli dnaE, polB, umuC, umuD, dinB, and mutS genes were (the UmuDЈ UmuC complex) and the UmuD subunit has been 2 amplified by PCR and fused in-frame to the LexA binding shown to bind to ␤ (9). domain through a Gly-Ala-Gly or Ala-Gly-Ala linker. Interac- Experimental evidence shows that at least some ␤-binding ␤ ␤ tions between and various LexA fusion proteins were tested in proteins can interact productively with from heterologous yeast EGY48 containing a lacZ reporter gene (EGY48p80p- species. For example, PolC subunits from Staphylococcus aureus, lacZ). Cotransformants were plated in synthetic complete me- Streptococcus pyogenes, and Bacillus subtilis can use E. coli ␤ as their processivity subunit (1, 10, 11). In contrast, E. coli DnaE ␤ cannot use from the other species (11), the E. coli clamp loader Abbreviations: PCNA, proliferating cell nuclear antigen; RT, room temperature. ␤ complex cannot load S. aureus (11), and the S. pyogenes clamp *B.P.D., K.K., and G.W. contributed equally to this work. ␤ loader complex cannot load E. coli (1). § ␤ To whom reprint requests should be addressed. E-mail: [email protected]. In the absence of any experimentally identified -binding sites The publication costs of this article were defrayed in part by page charge payment. This in proteins, a bioinformatics approach was undertaken to iden- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. tify putative ␤-binding motifs. The role of the putative motif was §1734 solely to indicate this fact. www.pnas.org͞cgi͞doi͞10.1073͞pnas.191384398 PNAS ͉ September 25, 2001 ͉ vol. 98 ͉ no. 20 ͉ 11627–11632 dium lacking appropriate supplements to maintain plasmid eubacterial PolB family is most similar to the archaeal PolB selection and patched onto indicator medium (SG͞Gal͞Raf͞- family, members of which use PCNA (15) [the eukaryotic͞ His͞-Leu͞-Trp͞-Ura with 5-bromo-4-chloro-3-indolyl ␤-D- archaeal equivalent of ␤ (16)]. The archaeal proteins contain a galactoside), grown at 30°C, and checked after 36 h for devel- peptide matching the consensus PCNA-binding motif (17) Qxx- opment of blue color. Results were compared with positive LxxFF (where x is any amino acid) at their carboxyl termini (15). (pLexA-53 with pB42AD-T) and negative (pLexA-Lam with We identified a pentapeptide with the consensus QLsLF (where pB42AD-T) controls grown in parallel. Cells also were grown to s is a small amino acid) at, or close to, the carboxyl termini of the midlog phase in selective media containing glucose or galactose. known eubacterial PolB proteins (Fig. 1). The similarity of this ␤-Galactosidase activity is expressed in Miller units. All results sequence to the PCNA-binding motif suggested that it may play are the mean of at least two independent assays with four a role in interaction with ␤. A similar conserved peptide replicates per assay. sequence at the carboxyl termini of members of the eubacterial PolC family also was identified (Fig. 1). The PolC and DnaE Binding Inhibition Assays. DNA polymerase III subunits ␣ and ␤ families contain homologous core domains, but have different were purified by modifications of published procedures using structural organizations and contain some unique domains (18). strains containing wild-type dnaEϩ and dnaNϩ derivatives of the However, a similar conserved peptide sequence was again ␭-promoter vector pCE30 (12). The ␦ subunit was prepared by identified in members of the DnaE family at the equivalent using strain BL21(DE3)͞pLysS͞pET␦, essentially as described location to that in PolC, rather than at the carboxyl termini (Fig. (13). The ␣ subunit at 20 ␮g͞ml was coated onto 96-well 1). The location of the peptide in E. coli ␣ is consistent with microtiter plates (Falcon flexible plates, Becton Dickinson) in previous work, which mapped a much larger region of ␤ binding ␮ ͞ 100 mM Na2CO3, pH 9.5 [50 l well, overnight at 4°C, or 4 h (19). Variants of the peptide sequence also were identified in at 25°C (room temperature, RT)]. Plates were washed in most eubacterial members of the UmuC and DinB1 families WB3 [20 mM Tris (pH 7.5), 0.1 mM EDTA, containing 0.05% (Fig. 1). However, the level of conservation of the proposed (vol͞vol) Tween 20]. This buffer was used in wash steps through- ␤-binding motifs in the DnaE and DinB1 families was signifi- out. Plates were then blocked with Blotto (5% skim milk powder cantly lower than in the PolB and PolC families. in WB3 100 ␮l͞well, RT) until required, then washed immedi- A large number of proteins that bind to PCNA and related ately before use. proteins has been identified (Table 2, which is published as The peptides were synthesized by fluorenylmethoxycarbonyl supporting information on the PNAS web site, www.pnas.org).
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