The Integron Integrase Efficiently Prevents the Melting Effect of Escherichia coli Single-Stranded DNA-Binding Protein on Folded attC Sites

Integrons play a major role in the dissemination of antibiotic resistance genes among bacteria. Rearrangement of gene cassettes occurs by recombination between attI and attC sites, catalyzed by the integron integrase. Integron recombination uses an unconventional mechanism involving a folded single-stranded attC site. This site could be a target for several host factors and more precisely for proteins able to bind single-stranded DNA. One of these, Escherichia coli single-stranded DNA-binding protein (SSB), regulates many DNA processes. We studied the influence of this protein on integron recombination. Our results show the ability of SSB to strongly bind folded attC sites and to destabilize them. This effect was observed only in the absence of the integrase. Indeed, we provided evidence that the integrase is able to counterbalance the observed effect of SSB on attC site folding. We showed that IntI1 possesses an intrinsic property to capture attC sites at the moment of their extrusion, stabilizing them and recombining them efficiently. The stability of DNA secondary structures in the chromosome must be restrained to avoid genetic instability (mutations or deletions) and/or toxicity (replication arrest). SSB, which hampers attC site folding in the absence of the integrase, likely plays an important role in maintaining the integrity and thus the recombinogenic functionality of the integron attC sites. We also tested the RecA host factor and excluded any role of this protein in integron recombination.     

Point Mutations in the Integron Integrase IntI1 That Affect Recombination and/or Substrate Recognition

The site-specific recombinase IntI1 found in class 1 integrons catalyzes the excision and integration of mobile gene cassettes, especially antibiotic resistance gene cassettes, with a site-specific recombination system. The integron integrase belongs to the tyrosine recombinase (phage integrase) family. The members of this family, exemplified by the lambda integrase, do not share extensive amino acid identities, but three invariant residues are found within two regions, designated box I and box II. Two conserved residues are arginines, one located in box I and one in box II, while the other conserved residue is a tyrosine located at the C terminus of box II. We have analyzed the properties of IntI1 variants carrying point mutations at the three conserved residues of the family in in vivo recombination and in vitro substrate binding. We have made four proteins with mutations of the conserved box I arginine (R146) and three mutants with changes of the box II arginine (R280); of these, MBP-IntI1(R146K) and MBP-IntI1(R280K) bind to the attI1 site in vitro, but only MBP-IntI1(R280K) is able to excise cassettes in vivo. However, the efficiency of recombination and DNA binding for MBP-IntI1(R280K) is lower than that obtained with the wild-type MBP-IntI1. We have also made two proteins with mutations of the tyrosine residue (Y312), and both mutant proteins are similar to the wild-type fusion protein in their DNA-binding capacity but are unable to catalyze in vivo recombination.Integrons are DNA elements that capture genes, especially antibiotic resistance genes, by a site-specific recombination system (32). The recombination system consists of a DNA integrase (Int) and two types of recombination sites, attI and attC (59-base element). The integrase gene (int) is located in the 5′ conserved segment of the integron structure (Fig. ​(Fig.1)1) and is a member of the tyrosine recombinase family (1, 4, 13, 23, 24). Three types of integrases, sharing around 50% identity among themselves, have been identified; they define integron classes 1, 2, and 3 (30). The 5′ conserved segment found in class 1 integrons also contains a promoter region responsible for the expression of inserted cassettes (11, 21) and the recombination site attI1 (31). The 3′ conserved segment of the class 1 integrons includes an ethidium bromide resistance determinant (qacEΔ1), a sulfonamide resistance gene (sulI), an open reading frame (ORF5) of unknown function, and further sequences that differ from one integron to another (5, 6, 28). The 3′ conserved segment of class 2 integrons includes transposition genes (20) while that of class 3 integrons has not yet been studied (2). The variable region, located between the two conserved segments, usually contains antibiotic resistance genes; In0 contains no inserted genes while In21 possesses eight cassettes with ten genes (or ORFs) in this region (5, 16). These genes are part of mobile cassettes which include a recombination site, attC, that differs from one gene to another (18, 33). Incoming genes must be associated with an attC to be recognized by the integron integrase and are preferentially inserted at the recombination site attI1 (11). Cassettes are excised as circular intermediates and integrated at core sites by the action of the integrase (8–10). The core site, defined as GTTRRRY, makes up the 3′ end of attI1 and attC, with the crossover taking place between the G and the first T (19). Antibiotic selection pressure can reveal cassette rearrangements in which a given resistance is nearest the promoter and thus most strongly expressed (10). Open in a separate windowFIG. 1General structure of class 1 integrons. Cassettes are inserted in the integron variable region by a site-specific recombination mechanism. The attI1 site is shown by a black circle, core sites are represented by ovals, the attC site is indicated by a black rectangle, and promoters are denoted by P. intIl, integrase gene; qacEΔ1, antiseptic resistance gene; sulI, sulfonamide resistance gene; orf5, gene of unknown function.Site-specific recombination, unlike homologous recombination, is characterized by relatively short, specific DNA sequences and requires only limited homology of the recombining partners (12). Site-specific recombination is an entirely conservative process since all DNA strands that are broken (two per exchange site) are rejoined in a process that involves neither ATP nor DNA synthesis. Homology alignments of site-specific recombinases assign them to two families: the resolvase family, named after the TnpR proteins encoded by the transposons γδ and Tn3, and the integrase family. The integrase family includes over 140 members to date, but they are highly diversified proteins (13, 23). Members of this family, which include the well-studied λ integrase, recombine DNA duplexes by executing two consecutive strand breakage and rejoining steps and a topoisomerization of the reactants. The first pair of exchanges form a four-way Holliday junction and the second pair resolve the junction to complete the recombination. The integrase nucleophile is a conserved tyrosine that becomes associated with a phosphate group on DNA. The cleavage sites on each DNA duplex are separated by 6 to 8 bp with a 5′ stagger, and the tyrosine joins to the 3′ phosphate (17).The initial definition of the integrase family was based on comparisons of seven sequences, and three invariant residues were identified: an HXXR cluster and a Y residue (4). Alignment of 28 sequences identified a fourth invariant position, occupied by an arginine residue (1). These four conserved residues are found in two boxes located in the second half of the protein. A recent analysis has shown that the conserved histidine is present in 136 of the 147 members (93%); this residue is then not conserved in all members of the family (13). Another recent analysis has identified three patches of residues located around box I, which seem to be important in the secondary structure of these proteins (23). In this study, we analyzed the properties of several mutants of the conserved residues R146, R280, and Y312 of the integron integrase IntI1 in in vivo recombination and in vitro substrate binding.

Construction of plasmids overexpressing mutant MBP-IntI1 fusion proteins.

The plasmids encoding various mutants of MBP-IntI1 were constructed by PCR using pLQ369 (50 ng) as a template (15). Two primer pairs, designed with the OLIGO software package (version 4.1; National Biosciences, Plymouth, Minn.), were used to construct each set of mutants. The R146 mutants were constructed with an XcmI-BamHI primer pair [IntI1(R146)-XcmI, 5′-TTCACCAGCTTCTGTATGGAACGGGCATG(A/G)(A/T)AATCAG-3′; IntI1(R146)-BamHI, 5′-CCGGATCCCTACCTCTCACT-3′], the R280 mutants were constructed with an NruI-XmnI primer pair [IntI1(R280)-NruI, 5′-AGCCGTCGCGAACGAGTGC(C/T)(C/T)GAGGG-3′; IntI1(R280)-XmnI, 5′-ACCCCTAATGAAGTGGTTCGTATCC-3′], and the Y312 mutants were constructed with a AatII-ScaI primer pair [IntI1(Y312)-AatII, 5′-ATTCCGACGTCTCTACTACGATGATTT(C/T)CACGC-3′; pLQ369-ScaI, 5′-ATGCTTTTCTGTGACTGGTG-3′] (restriction sites within primer sequences are underlined). PCR conditions were 10 min at 94°C, three cycles consisting of 45 s at 94°C, 45 s at 47°C, and 90 s at 72°C, 30 cycles consisting of 45 s at 94°C, 45 s at 60°C (50°C for Y312 mutants), and 90 s at 72°C, and a final elongation step of 10 min at 72°C. The XcmI, NruI, and AatII primers were degenerate in one or two positions, so that a single primer could give all mutants. Mutant PCR fragments were digested and cloned directly into pLQ369 digested with the same enzymes, except for the R146 mutant fragments that were subcloned into pLQ364 at first. New mutant PCR fragments were then amplified on these subclones, using IntI1(R146)-BamHI and IntI1(R280)-XmnI primers. These mutant PCR fragments were cleaved with BamHI and XmnI, and the resulting fragments were cloned into pLQ369. This avoids the necessity of partial digestion of pLQ369 with XcmI. Mutant clones were digested with restriction endonucleases and sequenced to determine the mutation.

In vivo recombination.

Mutant MBP-IntI1 clones were introduced into Escherichia coli TB1 {F′ araΔ(lac-proAB) rpsL (Str^r) [φ80dlacΔ(lacZ)M15] hsdR(r_K⁻m_K⁻)} containing pLQ428 by transformation (Fig. ​(Fig.22 and Table ​Table1).1). E. coli TB1 cells containing pLQ428 and one of the MBP-IntI1 mutants were grown at 37°C for 3 h in Luria-Bertani medium. Excision of the aacA1-ORFG and/or ORFH cassettes was induced by the overexpression of the malE-intI1 gene by using 0.3 mM isopropyl-β-d-thiogalactopyranoside (IPTG; Sigma Chemical Co.) and by incubation at 37°C for another 3 h. Cell culture was done in the presence of 50 μg of ampicillin per ml, 15 μg of amikacin per ml, and 50 μg of chloramphenicol per ml. Plasmid DNA was then prepared from 5-ml cultures with the Perfect Prep DNA extraction kit (Mandel Corporation). In order to determine the capacity of mutant MBP-IntI1 proteins to excise aacA1-ORFG and/or ORFH cassettes of In21, we used PCR primers pACYC184-5′ (5′-TGTAGCACCTGAAGTCAGCC-3′) and pACYC184-3′ (5′-ATACCCACGCCGAAACAAG-3′) (Fig. ​(Fig.2,2, primers 1 and 2) to detect the reduction of pLQ428 length. PCR conditions were 10 min at 94°C, 30 cycles consisting of 1 min at 94°C, 1 min at 60°C, and 5 min at 72°C, and a final elongation step of 10 min at 72°C. A major PCR fragment can be seen in each lane containing a DNA preparation from a mutant clone (Fig. ​(Fig.3,3, lanes 2 to 9). This band is 2,499 bp long and, as determined by restriction enzyme digestions, represents the pLQ428 clone without any cassette excision (data not shown). This band is also observed in the negative control, which is the pMAL-c2 vector without any gene fused to malE (Fig. ​(Fig.3,3, lane 12). Open in a separate windowFIG. 2Representation of plasmids used in this study. The positions of the three invariant residues of the integrase family are indicated, along with restriction sites used to construct mutant proteins. Core sites are represented by black circles, and attCs are shown by white boxes. The numbered arrows represent the PCR primers used to detect excision events, pACYC184-5′ (1) and pACYC184-3′ (2). bla, gene encoding β-lactamase; cat, gene encoding chloramphenicol acetyltransferase; intIl, gene encoding the integron integrase (IntI1); malE, gene encoding the maltose binding protein (MBP); ori, origin of replication; P_tac, tac promoter; P_tet, tetracycline promoter. Only relevant restriction sites are indicated.

TABLE 1

Plasmids used in this study

Structural Features of Single-Stranded Integron Cassette attC Sites and Their Role in Strand Selection

We recently showed that cassette integration and deletion in integron platforms were occurring through unconventional site-specific recombination reactions involving only the bottom strand of attC sites. The lack of sequence conservation among attC sites led us to hypothesize that sequence-independent structural recognition determinants must exist within attC sites. The structural data obtained from a synaptic complex of the Vibrio cholerae integrase with the bottom strand of an attC site has shown the importance of extra helical bases (EHB) inside the stem-loop structure formed from the bottom strand. Here, we systematically determined the contribution of three structural elements common to all known single-stranded attC site recombination substrates (the EHBs, the unpaired central spacer (UCS), and the variable terminal structure (VTS)) to strand choice and recombination. Their roles have been evaluated in vivo in the attI×attC reaction context using the suicide conjugation assay we previously developed, but also in an attC×attC reaction using a deletion assay. Conjugation was used to deliver the attC sites in single-stranded form. Our results show that strand choice is primarily directed by the first EHB, but the presence of the two other EHBs also serves to increase this strand selection. We found that the structure of the central spacer is essential to achieve high level recombination of the bottom strand, suggesting a dual role for this structure in active site exclusion and for hindering the reverse reaction after the first strand exchange. Moreover, we have shown that the VTS has apparently no role in strand selectivity.     

Identification of key structural determinants of the IntI1 integron integrase that influence attC x attI1 recombination efficiency

The integron platform codes for an integrase (IntI) from the tyrosine family of recombinases that mediates recombination between a proximal double-strand recombination site, attI and a single-strand target recombination site, attC. The attI site is only recognized by its cognate integrase, while the various tested attCs sites are recombined by several different IntI integrases. We have developed a genetic system to enrich and select mutants of IntI1 that provide a higher yield of recombination in order to identify key protein structural elements important for attC × attI1 recombination. We isolated mutants with higher activity on wild type and mutant attC sites. Interestingly, three out of four characterized IntI1 mutants selected on different substrates are mutants of the conserved aspartic acid in position 161. The IntI1 model we made based on the VchIntIA 3D structure suggests that substitution at this position, which plays a central role in multimer assembly, can increase or decrease the stability of the complex and accordingly influence the rate of attI × attC recombination versus attC × attC. These results suggest that there is a balance between the specificity of the protein and the protein/protein interactions in the recombination synapse.     

Human Origin Recognition Complex Binds Preferentially to G-quadruplex-preferable RNA and Single-stranded DNA

Origin recognition complex (ORC), consisting of six subunits ORC1–6, is known to bind to replication origins and function in the initiation of DNA replication in eukaryotic cells. In contrast to the fact that Saccharomyces cerevisiae ORC recognizes the replication origin in a sequence-specific manner, metazoan ORC has not exhibited strict sequence-specificity for DNA binding. Here we report that human ORC binds preferentially to G-quadruplex (G4)-preferable G-rich RNA or single-stranded DNA (ssDNA). We mapped the G-rich RNA-binding domain in the ORC1 subunit, in a region adjacent to its ATPase domain. This domain itself has an ability to preferentially recognize G4-preferable sequences of ssDNA. Furthermore, we found, by structure modeling, that the G-rich RNA-binding domain is similar to the N-terminal portion of AdoMet_MTase domain of mammalian DNA methyltransferase 1. Therefore, in contrast with the binding to double-stranded DNA, human ORC has an apparent sequence preference with respect to its RNA/ssDNA binding. Interestingly, this specificity coincides with the common signature present in most of the human replication origins. We expect that our findings provide new insights into the regulations of function and chromatin binding of metazoan ORCs.     

Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form,and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site

The P9-1 protein of Rice black-streaked dwarf virus (RBSDV) is an essential part of the viroplasm. However, little is known about its nature or biological function in the viroplasm. In this study, the structure and function of P9-1 were analyzed for in vitro binding to nucleic acids. We found that the P9-1 protein preferentially bound to single-stranded versus double-stranded nucleic acids; however, the protein displayed no preference for RBSDV versus non-RBSDV single-stranded ssRNA (ssRNA). A gel mobility shift assay revealed that the RNA gradually shifted as increasing amounts of P9-1 were added, suggesting that multiple subunits of P9-1 bind to ssRNA. By using discontinuous blue native gel and chromatography analysis, we found that the P9-1 protein was capable of forming dimers, tetramers, and octamers. Strikingly, we demonstrated that P9-1 preferentially bound to ssRNA in the octamer, rather than the dimer, form. Deletion of the C-terminal arm resulted in P9-1 no longer forming octamers; consequently, the deletion mutant protein bound to ssRNA with significantly lower affinity and with fewer copies bound per ssRNA. Alanine substitution analysis revealed that electropositive amino acids among residues 25 to 44 are important for RNA binding and map to the central interior structure that was formed only by P9-1 octamers. Collectively, our findings provide novel insights into the structure and function of RBSDV viroplasm protein P9-1 binding to RNA.     

An Unusual Helix Turn Helix Motif in the Catalytic Core of HIV-1 Integrase Binds Viral DNA and LEDGF

Background

Integrase (IN) of the type 1 human immunodeficiency virus (HIV-1) catalyzes the integration of viral DNA into host cellular DNA. We identified a bi-helix motif (residues 149–186) in the crystal structure of the catalytic core (CC) of the IN-Phe185Lys variant that consists of the α₄ and α₅ helices connected by a 3 to 5-residue turn. The motif is embedded in a large array of interactions that stabilize the monomer and the dimer.

Principal Findings

We describe the conformational and binding properties of the corresponding synthetic peptide. This displays features of the protein motif structure thanks to the mutual intramolecular interactions of the α₄ and α₅ helices that maintain the fold. The main properties are the binding to: 1- the processing-attachment site at the LTR (long terminal repeat) ends of virus DNA with a K_d (dissociation constant) in the sub-micromolar range; 2- the whole IN enzyme; and 3- the IN binding domain (IBD) but not the IBD-Asp366Asn variant of LEDGF (lens epidermal derived growth factor) lacking the essential Asp366 residue. In our motif, in contrast to the conventional HTH (helix-turn-helix), it is the N terminal helix (α₄) which has the role of DNA recognition helix, while the C terminal helix (α₅) would rather contribute to the motif stabilization by interactions with the α₄ helix.

Conclusion

The motif, termed HTHi (i, for inverted) emerges as a central piece of the IN structure and function. It could therefore represent an attractive target in the search for inhibitors working at the DNA-IN, IN-IN and IN-LEDGF interfaces.     

DNA complexes obtained with the integron integrase IntI1 at the attI1 site.

Integrons are genetic elements that are able to capture genes by a site-specific recombination mechanism. Integrons contain a gene coding for a lambda-like integrase that carries out site-specific recombination by interacting with two different target sites; the attI site and the palindromic sequence attC (59 base element). Cassette integrations usually involve the attI site, while cassette excisions use attC . Therefore, the integrase should bind both sites to cleave DNA and perform site-specific recombination reactions. We have used purified maltose-binding protein fused with the integrase (MBP-IntI1) and native IntI1 protein and gel retardation assays with fragments containing the complete and partial attI1 site to show formation of four complexes in this region. Chemical modification of specific nucleotides within the attI1 site was used to investigate their interference with binding of the integrase protein. We attribute IntI1 specific binding to four regions in the attI1 site and a GTTA consensus sequence is found in three of the four regions. Interference by modified guanine and thymine residues in the DNA major groove and adenine residues in the minor groove were observed, indicating that the integrase interacts with both sides of the helix. Binding of IntI1 to attC is also discussed.     

Retroviral Integrase Proteins and HIV-1 DNA Integration

Retroviral integrases catalyze two reactions, 3′-processing of viral DNA ends, followed by integration of the processed ends into chromosomal DNA. X-ray crystal structures of integrase-DNA complexes from prototype foamy virus, a member of the Spumavirus genus of Retroviridae, have revealed the structural basis of integration and how clinically relevant integrase strand transfer inhibitors work. Underscoring the translational potential of targeting virus-host interactions, small molecules that bind at the host factor lens epithelium-derived growth factor/p75-binding site on HIV-1 integrase promote dimerization and inhibit integrase-viral DNA assembly and catalysis. Here, we review recent advances in our knowledge of HIV-1 DNA integration, as well as future research directions.     

CTnDOT Integrase Interactions with Attachment Site DNA and Control of Directionality of the Recombination Reaction

IntDOT is a tyrosine recombinase encoded by the conjugative transposon CTnDOT. The core binding (CB) and catalytic (CAT) domains of IntDOT interact with core-type sites adjacent to the regions of strand exchange, while the N-terminal arm binding (N) domain interacts with arm-type sites distal to the core. Previous footprinting experiments identified five arm-type sites, but how the arm-type sites participate in the integration and excision of CTnDOT was not known. In vitro integration assays with substrates containing arm-type site mutants demonstrated that attDOT sequences containing mutations in the L1 arm-type site or in the R1 and R2 or R1 and R2′ arm-type sites were dramatically defective in integration. Substrates containing mutations in the L1 and R1 arm-type sites showed a 10- to 20-fold decrease in detectable in vitro excision, but introduction of multiple arm-type site mutations in attR did not have an effect on the excision frequency. A sixth arm-type site, the R1′ site, was also identified and shown to be required for integration and important for efficient excision. These results suggest that intramolecular IntDOT interactions are required for integration, while the actions of accessory factors are more important for excision. Gel shift assays performed in the presence of core- and arm-type site DNAs showed that IntDOT affinity for the attDOT core was enhanced when IntDOT was simultaneously bound to arm-type site DNA.Conjugative transposons (CTns), also known as integrative and conjugative elements (ICEs), are mobile genetic elements that are widespread in Bacteroides spp. and are implicated in the spread of antibiotic resistance. These elements are normally integrated into the host chromosome but can excise, replicate, and transfer to a recipient cell by conjugation (34). Since CTns commonly carry antibiotic resistance genes, it is likely that the increase in antibiotic-resistant Bacteroides strains has been mediated through the lateral transfer of these elements (36). One of the best-studied ICEs in Bacteroides is the conjugative transposon CTnDOT. CTnDOT is 65 kb in size and carries genes encoding resistance to tetracycline and erythromycin. Over the past 30 years, the incidence of tetracycline resistance has increased to 80% of Bacteroides isolates due to the presence of CTnDOT-type elements (36).Integration and excision of CTnDOT results from site-specific recombination between regions of DNA known as attachment (att) sites. During integration, the joined ends of the closed circular intermediate (attDOT) recombine with the bacterial target sequence (attB) to form the recombinant sites (attL and attR). The integration reaction requires IntDOT, a CTnDOT-encoded protein that has been identified as a member of the tyrosine recombinase family, as well as a host factor encoded by Bacteroides (8, 21). Site-specific recombination between the attL and attR attachment sites results in excision of CTnDOT from the host chromosome. IntDOT is also required for excision, as are three element-encoded proteins: Orf2c, Orf2d, and Exc, as well a Bacteroides host factor (8, 38). The roles of these accessory proteins are not well understood, although Orf2c and Orf2d have been shown to bind DNA (unpublished results).One of the best-studied tyrosine recombinases is the integrase (Int) of the lambda system. The C terminus of Int includes the core binding (CB) and catalytic (CAT) domains that bind to core-type sites, which flank the sites of cleavage and strand exchange (2, 24). The N-terminal arm-binding (N) domain binds to arm-type sites that are distal to the core-type sites. In the presence of the appropriate host and accessory factors, Int binding to arm-type sites is required for the formation of higher-order protein/DNA complexes known as intasomes, which are required for integration and excision (15, 18, 22). Int is capable of making intramolecular interactions (interactions between Int monomers on the same attachment site) and intermolecular interactions (interactions between Int monomers on different attachment sites) during recombination (15, 16). In the lambda system, the directionality of the reaction is regulated by Int interactions with arm-type sites in conjunction with the integration host factor (IHF) during the formation of an integrative intasome, or IHF, Xis, and FIS during the formation of the two excisive intasomes (1, 4, 42).Presumably, IntDOT occupancy of specific arm-type sites in conjunction with interactions of accessory factors with att sites leads to the assembly of integrative or excisive intasomes and thus contributes to the directionality of IntDOT-mediated recombination. Previous DNase I footprinting experiments identified five arm-type binding sites on attDOT (11). In this study, mutations were constructed in the five sites to determine their roles in the integration and excision of CTnDOT. In addition, a sixth arm-type site was discovered that is important for both integrative and excisive recombination. The results of gel shift assays also show that the interaction of IntDOT with core-type sites and arm-type sites involves cooperative interactions.     

Gbp1p, a Protein with RNA Recognition Motifs, Binds Single-Stranded Telomeric DNA and Changes Its Binding Specificity upon Dimerization

Gbp1p is a putative telomere-binding protein from Chlamydomonas reinhardtii that contains two RNA recognition motifs (RRMs) which are commonly found in heterogeneous nuclear ribonucleoproteins (hnRNPs). Previously we demonstrated that Gbp1p binds single-stranded DNA (ssDNA) containing the Chlamydomonas telomeric sequence but not the RNA containing the cognate sequence. Here we show that at lower protein concentrations Gbp1 can also bind an RNA containing the cognate sequence. We found that mutation of the two RRM motifs of Gbp1p to match the highly conserved region of hnRNP RRMs did not alter the affinity of Gbp1p for either RNA or DNA. The ability of Gbp1p to associate with either of these two nucleic acids is governed by the dimerization state of the protein. Monomeric Gbp1p associates with either ssDNA or RNA, showing a small binding preference for RNA. Dimeric Gbp1p has a strong preference for binding ssDNA and shows little affinity for RNA. To the best of our knowledge, this is the first example of a protein that qualitatively shifts its nucleic acid binding preference upon dimerization. The biological implications of a telomere-binding protein that is regulated by dimerization are discussed.     

Hedgehog Pathway Antagonist 5E1 Binds Hedgehog at the Pseudo-active Site

Proper hedgehog (Hh) signaling is crucial for embryogenesis and tissue regeneration. Dysregulation of this pathway is associated with several types of cancer. The monoclonal antibody 5E1 is a Hh pathway inhibitor that has been extensively used to elucidate vertebrate Hh biology due to its ability to block binding of the three mammalian Hh homologs to the receptor, Patched1 (Ptc1). Here, we engineered a murine:human chimeric 5E1 (ch5E1) with similar Hh-binding properties to the original murine antibody. Using biochemical, biophysical, and x-ray crystallographic studies, we show that, like the regulatory receptors Cdon and Hedgehog-interacting protein (Hhip), ch5E1 binding to Sonic hedgehog (Shh) is enhanced by calcium ions. In the presence of calcium and zinc ions, the ch5E1 binding affinity increases 10–20-fold to tighter than 1 nm primarily because of a decrease in the dissociation rate. The co-crystal structure of Shh bound to the Fab fragment of ch5E1 reveals that 5E1 binds at the pseudo-active site groove of Shh with an epitope that largely overlaps with the binding site of its natural receptor antagonist Hhip. Unlike Hhip, the side chains of 5E1 do not directly coordinate the Zn²⁺ cation in the pseudo-active site, despite the modest zinc-dependent increase in 5E1 affinity for Shh. Furthermore, to our knowledge, the ch5E1 Fab-Shh complex represents the first structure of an inhibitor antibody bound to a metalloprotease fold.     

Defining the DNA Substrate Binding Sites on HIV-1 Integrase

A tetramer model for human immunodeficiency virus type 1 (HIV-1) integrase (IN) with DNA representing long terminal repeat (LTR) termini was previously assembled to predict the IN residues that interact with the LTR termini; these predictions were experimentally verified for nine amino acid residues [Chen, A., Weber, I. T., Harrison, R. W. & Leis, J. (2006). Identification of amino acids in HIV-1 and avian sarcoma virus integrase subsites required for specific recognition of the long terminal repeat ends. J. Biol. Chem., 281, 4173-4182]. In a similar strategy, the unique amino acids found in avian sarcoma virus IN, rather than HIV-1 or Mason-Pfizer monkey virus IN, were substituted into the structurally related positions of HIV-1 IN. Substitutions of six additional residues (Q44, L68, E69, D229, S230, and D253) showed changes in the 3′ processing specificity of the enzyme, verifying their predicted interaction with the LTR DNA. The newly identified residues extend interactions along a 16-bp length of the LTR termini and are consistent with known LTR DNA/HIV-1 IN cross-links. The tetramer model for HIV-1 IN with LTR termini was modified to include two IN binding domains for lens-epithelium-derived growth factor/p75. The target DNA was predicted to bind in a surface trench perpendicular to the plane of the LTR DNA binding sites of HIV-1 IN and extending alongside lens-epithelium-derived growth factor. This hypothesis is supported by the in vitro activity phenotype of HIV-1 IN mutant, with a K219S substitution showing loss in strand transfer activity while maintaining 3′ processing on an HIV-1 substrate. Mutations at seven other residues reported in the literature have the same phenotype, and all eight residues align along the length of the putative target DNA binding trench.     

Intracellular Steady-State Concentration of Integron Recombination Products Varies with Integrase Level and Growth Phase

IntI1 mediates the recombination of antibiotic-resistant gene cassettes between different integrons in the same cell, facilitating the persistence and dissemination of these genes. Historically, integrase activity has been measured by conjugating recombinant products from donor cells overexpressing integrase and quantifying them in recipient cells. Here we report the first measurements of the steady-state intracellular abundance of integrase-mediated recombination products in strains expressing natural or high IntI1 levels. Recombination products in both high and natural integrase strains increased markedly through late log phase and continued to rise in stationary phase in the high integrase strain, but declined in the natural expression strain. Simple acquisition of gene cassettes was seen only in strains expressing high integrase; in strains with natural integrase levels, only cointegrates between the two integron-bearing plasmids were detectable at all growth stages. Unexpectedly, more attI × attI than attC × attI recombination products were seen in log phase for both strains; however, in stationary phase, the high integrase strain increased attC recombination, consistent with earlier observations of integrase crossover site preferences. Thus, direct quantification of the steady-state concentration of recombination products reveals that the integrase's intracellular concentration affects the amount and type of recombination events in a growth-phase-dependent manner.     

The Saccharomyces cerevisiae Mlh1-Mlh3 Heterodimer Is an Endonuclease That Preferentially Binds to Holliday Junctions

MutLγ, a heterodimer of the MutL homologues Mlh1 and Mlh3, plays a critical role during meiotic homologous recombination. The meiotic function of Mlh3 is fully dependent on the integrity of a putative nuclease motif DQHAX₂EX₄E, inferring that the anticipated nuclease activity of Mlh1-Mlh3 is involved in the processing of joint molecules to generate crossover recombination products. Although a vast body of genetic and cell biological data regarding Mlh1-Mlh3 is available, mechanistic insights into its function have been lacking due to the unavailability of the recombinant protein complex. Here we expressed the yeast Mlh1-Mlh3 heterodimer and purified it into near homogeneity. We show that recombinant MutLγ is a nuclease that nicks double-stranded DNA. We demonstrate that MutLγ binds DNA with a high affinity and shows a marked preference for Holliday junctions. We also expressed the human MLH1-MLH3 complex and show that preferential binding to Holliday junctions is a conserved capacity of eukaryotic MutLγ complexes. Specific DNA recognition has never been observed with any other eukaryotic MutL homologue. MutLγ thus represents a new paradigm for the function of the eukaryotic MutL protein family. We provide insights into the mode of Holliday junction recognition and show that Mlh1-Mlh3 prefers to bind the open unstacked Holliday junction form. This further supports the model where MutLγ is part of a complex acting on joint molecules to generate crossovers in meiosis.     

Analysis by Mutagenesis of a Chromosomal Integron Integrase from Shewanella amazonensis SB2BT

Integrons are mobile genetic elements that can integrate and disseminate genes as cassettes by a site-specific recombination mechanism. Integrons contain an integrase gene (intI) that carries out recombination by interacting with two different target sites; the attI site in cis with the integrase and the palindromic attC site of a cassette. The plasmid-specified IntI1 excises a greater variety of cassettes (principally antibiotic resistance genes), and has greater activity, than chromosomal integrases. The aim of this study was to analyze the capacity of the chromosomal integron integrase SamIntIA of the environmental bacterium Shewanella amazonensis SB2BT to excise various cassettes and to compare the properties of the wild type with those of mutants that substitute consensus residues of active integron integrases. We show that the SamIntIA integrase is very weakly active in the excision of various cassettes but that the V206R, V206K, and V206H substitutions increase its efficiency for the excision of cassettes. Our results also suggest that the cysteine residue in the β-5 strand is essential to the activity of Shewanella-type integrases, while the cysteine in the β-4 strand is less important for the excision activity.Integrons are genetic elements that capture and rearrange genes that are contained within mobile gene cassettes by a mechanism of site-specific recombination mediated by an integrase (3). Several types of integron integrases have been described for clinical and environmental bacteria; classes 1, 2, and 3 integron integrases (1, 10, 11) and VchIntIA (17) and IntI9 (12) integrases are the only ones that are associated with antibiotic resistance genes. Some of these integrases were found exclusively on plasmids (IntI2*) (11) or on chromosomes (VchIntIA) (17), while others were found in both genetic contexts (IntI1) (7, 8, 20, 21). The efficiency of integron integrases to carry out cassette excision varies from one integrase to another and also depends on the structure and sequence of the attC sites located at both ends of the gene. IntI1 is generally the most active integrase, followed by IntI3. IntI2*179E and SonIntIA are less active but appear to tolerate more variation in attC sites. These enzymes could serve as models for determining important residues responsible for high levels of activity, using mutagenesis to substitute consensus residues and assaying for gain of function.Class 1 integrons, carrying the intI1 integrase gene, are generally associated with mobile elements, such as plasmids and Tn21-like transposons, and are most frequently found in clinical isolates (18). They are found mainly among gram-negative bacteria and especially among enterobacteria and pseudomonads (14). Class 1 integrons have also been found in some gram-positive bacteria, such as Enterococcus, Staphylococcus, and Corynebacterium (6). The clinical-type class 1 integrons (7) consist of two conserved regions and a variable region in which resistance genes are inserted in the form of cassettes (Fig. ​(Fig.1A).1A). These integrons were clearly derived from a structure related to Tn402, as they share many characteristics associated with this type of transposon (21). The common ancestor of clinical-type class 1 integrons was possibly a member of an integron pool that was acquired by diverse Betaproteobacteria (7). This hypothesis is based on the recent isolation of several new class 1 integron integrases from environmental DNA samples which are not associated with antibiotic resistance genes or with Tn402-like transposons (7, 8, 21).Open in a separate windowFIG. 1.(A) General structure of clinical-type class 1 integrons. Cassettes are inserted in the variable region of integrons by a site-specific recombinational mechanism. The attI1 and attC sites are shown by tiling and diagonal black lines, respectively, and promoters are denoted by P1, P2, P3, and P. Genes are as follows: intI1, integrase gene; qacEΔ1, antiseptic resistance gene; sul1, sulfonamide resistance gene; orf5, gene of unknown function. (B) Representation of the chromosomal integron of S. amazonensis SB2BT. The attI_Sam and attC sites are shown by a black box and horizontal black lines, respectively. Genes are as follows: SamintIA, integrase gene; orf, open reading frame gene.Class 2 integrons, carrying the intI2* integrase pseudogene, are present on Tn7 transposons and their derivatives (11). The intI2* gene encodes an integrase identical to 46% with IntI1, but its reading frame was interrupted by an early termination codon. The activity of this protein is restored when the stop codon at position 179 is replaced by a glutamate codon (11). Recently, two new intI2 genes were identified within integrons found in Providencia stuartii (2) and Escherichia coli (16). The sequences of these genes are not interrupted; position 179 is occupied by a glutamine codon, and the genes apparently code for functional enzymes. These intI2 genes each differ from intI2* of Tn7 at five positions (2, 16).Class 3 integrons, characterized by the presence of the intI3 gene, have been found in Serratia marcescens AK9373, in Klebsiella pneumoniae FFUL 22K isolated in Portugal, in four strains of Pseudomonas putida isolated in Japan, and more recently, in Delftia acidovorans C17 and Delftia tsuruhatensis A90 (1, 4, 19, 23). The IntI3 integrase has 61% identity with IntI1.The class 4 integron, with VchintIA, is an integron carried by the small chromosome of Vibrio cholerae O:1 569B (17). This integron contains more than 216 open reading frames (ORFs) coding for proteins of unknown functions associated with V. cholerae repetitive DNA sequence (VCR) elements to form 179 cassettes, and occupies about 3% of the bacterial genome.In recent years, the draft genomes of various environmental strains led to the identification of more than 100 new integron integrases. Among these, the SonintIA and NeuintIA integrase genes have been found, respectively, in genomes of Shewanella oneidensis MR-1 and Nitrosomonas europaea and shown to be active in cassette excision and integration (5, 13). Shewanella amazonensis SB2BT is an environmental gram-negative gammaproteobacterium that plays an important role in the bioremediation of contaminated metals and radioactive wastes (22). The U.S. Department of Energy Joint Genome Institute sequenced its 4.3-Mbp genome (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP000507","term_id":"119765642","term_text":"CP000507"}}CP000507). The genome encodes an integron integrase, SamIntIA, which is 64.8% identical to SonIntIA and 60.2% identical to IntI2* but only 46.9% identical to VchIntIA and 44.6% to IntI1. A sequence alignment of SamIntIA, SonIntIA, and IntI2* indicates that they are closely related, especially in the N-terminal and the C-terminal regions.Several residues of SamIntIA differed from a consensus alignment of active integron integrases. We wished to determine whether SamIntIA is active, compare its activity to that of SonIntIA and of IntI2*179E, and determine whether the alteration of certain residues affects its excision activity.     

Binding of the purified integron DNA integrase IntI1 to integron- and cassette-associated recombination sites

The site-specific recombinase IntI1, encoded by class 1 integrons, catalyses the integration and excision of gene cassettes by recognizing two classes of sites, the integron-associated attI1 site and the 59-base element (59-be) family of sites that are associated with gene cassettes. IntI1 includes the four conserved amino acids that are characteristic of members of the integrase family, and IntI1 proteins with single amino acid substitutions at each of these positions had substantially reduced catalytic activity, consistent with this classification. IntI1 was purified as a fusion protein and shown to bind to isolated attI1 or 59-be recombination sites. Binding to attI1 was considerably stronger than to a 59-be. Binding adjacent to the recombination cross-over point was not detected. A strong IntI1 binding site within attI1 was localized by both deletion and footprinting analysis to a 14 bp region 24–37 bp to the left of the recombination cross-over point, and this region is known to be critical for recombination in vivo ( Recchia et al ., 1994 ). An imperfect (13/15) direct repeat of this region, located 41–55 bp to the left of the recombination cross-over point, contains a weaker IntI1 binding site. Mutation of the stronger binding site showed that a single base pair change accounted for the difference in the strength of binding.     

Interactions of HIV-1 DNA Heterocyclic Bases with Viral Integrase

Integrase (IN) is responsible for one of the key stages in the replication cycle of human immunodeficiency virus type 1, namely, integration of a DNA copy of the viral RNA into the infected cell genome. IN recognizes the nucleotide sequences located at the ends of the U3 and U5 regions of long terminal repeats (LTRs) of the viral DNA and sequentially catalyzes the 3-end processing and strand transfer reactions. Analogs of U5 regions containing non-nucleoside insertions have been used to study the interaction between IN and viral DNA. Substrate modification has been demonstrated to have almost no effect on the rate of DNA binding by IN. However, the removal of heterocyclic bases from positions 5 and 6 of the substrate molecule and from position 3 of the processed strand almost completely inhibits IN enzymatic activity, which indicates the importance of these bases for the formation of an active enzyme–substrate complex. By contrast, modification of the third base of the nonprocessed strand stimulates 3-processing. Since the base removal disturbs the complementary and stacking interactions in DNA, these results indicate that double-helix destabilization near the cleaved bond promotes 3-end processing.