ABSTRACT
Loop-out-type recombination is a type of intrachromosomal recombination followed by the excision of a chromosomal region. The detailed mechanism underlying this recombination and the genes involved in loop-out recombination remain unknown. In the present study, we investigated the functions of ku70, ligD, rad52, rad54, and rdh54 in the construction of large chromosomal deletions via loop-out recombination and the effect of the position of the targeted chromosomal region on the efficiency of loop-out recombination in Aspergillus oryzae. The efficiency of generation of large chromosomal deletions in the near-telomeric region of chromosome 3, including the aflatoxin gene cluster, was compared with that in the near-centromeric region of chromosome 8, including the tannase gene. In the Δku70 and Δku70-rdh54 strains, only precise loop-out recombination occurred in the near-telomeric region. In contrast, in the ΔligD, Δku70-rad52, and Δku70-rad54 strains, unintended chromosomal deletions by illegitimate loop-out recombination occurred in the near-telomeric region. In addition, large chromosomal deletions via loop-out recombination were efficiently achieved in the near-telomeric region, but barely achieved in the near-centromeric region, in the Δku70 strain. Induction of DNA double-strand breaks by I-SceI endonuclease facilitated large chromosomal deletions in the near-centromeric region. These results indicate that ligD, rad52, and rad54 play a role in the generation of large chromosomal deletions via precise loop-out-type recombination in the near-telomeric region and that loop-out recombination between distant sites is restricted in the near-centromeric region by chromosomal structure.
INTRODUCTION
Aspergillus oryzae and Aspergillus sojae are multinucleate filamentous fungi that are extensively used for industrial production of enzymes and fermented foods. The genome of the A. oryzae RIB40 strain has been sequenced and is available for genetic research (13). It is of scientific and industrial interest to improve the properties of a strain by generating a minimal genome construct via removal of chromosomal regions that are unnecessary for cell survival or enzyme production. In fact, such attempts have already been reported in prokaryotes (5, 11, 19). However, there have been few reports about generating minimal genome constructs in filamentous fungi. Previously, we established techniques for generating large chromosomal deletions by 2 recombination methods, namely, replacement-type recombination (Fig. 1A) and loop-out recombination (Fig. 1B), by using nonhomologous end joining-deficient strains of A. oryzae and A. sojae (23, 24). The replacement-type recombination involves the integration of exogenous DNA into chromosomes during a transformation process, such as the protoplast polyethylene glycol (PEG) method or electroporation (Fig. 1A). The integration of exogenous DNA into chromosomes can be understood as a phenomenon that occurs during the repair of DNA double-strand breaks (DSBs) in the chromosomes by the exogenous DNA fragment (2, 8). The basic mechanism of the replacement-type recombination is similar to that of the generally recognized DSB repair process, and the genes involved in the recombination have been well studied in the budding yeast Saccharomyces cerevisiae (20). When targeted integration of the exogenous DNA into the chromosome occurs by replacement-type recombination, genes related to homologous recombination, the rad52 epistasis group of genes, such as rad51, rad52, rad54, rdh54, MRE11, and XRS2, are reported to be important (20). rad51, rad52, and rad54 play major roles in homologous recombination. rdh54 is a rad54 homologue that functions in diploid-specific mitotic recombination (10). On the other hand, in the case of random integration of exogenous DNA into a chromosome, genes involved in nonhomologous end joining (NHEJ) such as ku70, ku80, lig4, and XRCC4 play important roles (4). The Ku protein, consisting of the Ku70-Ku80 heterodimer, binds to the end of DNA to activate the NHEJ pathway. lig4 encodes a specific ligase for the NHEJ pathway. Inhibition of the NHEJ-related genes ku70, ku80, and ligD (human lig4 homologue) remarkably increases targeting frequency in A. oryzae and A. sojae (15, 22) as well as in Neurospora strains (7, 16). Moreover, deficiency of nonhomologous end joining allows large chromosomal deletions by replacement-type recombination with high efficiency, even in the generation of a 470-kb chromosomal deletion in A. oryzae (24). In the case of loop-out recombination, the detailed mechanism and the genes involved are still poorly understood (Fig. 1B). Only rad51, rad52, rad54, rad55, and rad57 have been reported to play a role in recombination between direct repeat sequences (recombination between 300-bp lengths of long terminal repeat [LTR] sequence separated by 5-kb lengths of Ty element) in S. cerevisiae (12, 20). Construction of large chromosomal deletions by loop-out recombination was reported in Aspergillus oryzae, A. sojae, and the fission yeast Schizosaccharomyces pombe (6, 23). In the case of A. oryzae and A. sojae, 200-kb chromosomal deletions were efficiently achieved by loop-out recombination by using ku70-deficient strains (23). The large genomic deletions generated by loop-out recombination enabled the construction of multiple deletions in A. oryzae and A. sojae by marker recycling (22, 23). Through this recombination method, no additional sequences were observed in the resultant deletion strains; this is a feature of considerable value for the breeding of food-grade microorganisms (23). However, the frequency of generation of large chromosomal deletions via loop-out recombination is much lower than that via replacement-type recombination. In addition, loop-out recombination tended to occur less readily in proportion to the length of the deletion range and was also affected by the location of the deleted region in the chromosome (23). Loop-out recombination is thought to be more sensitive to the chromosome structure than is the replacement-type recombination (23, 24). Therefore, there is a need to establish a method to facilitate the generation of large chromosomal deletions by loop-out recombination in A. oryzae.
(A and B) Schematic representation of chromosome deletion by replacement-type recombination (A) and loop-out-type recombination (B). (C) The effect of a deficiency of recombination-related genes on the generation of a large chromosomal deletion by loop-out recombination was assessed by the construction of a targeted large chromosomal deletion, which included the AF biosynthetic gene cluster, by loop-out recombination in the near-telomeric region. C47, AO090026000047. (D) The effect of chromosomal location on the generation of a large chromosomal deletion by loop-out recombination was assessed by the construction of targeted chromosomal deletions, including 3-, 57-, 100-, and 200-kb regions, by loop-out recombination in the near-centromeric region.
For the reasons mentioned above, in this study, we tried to accomplish the following goals: (i) identification of genes related to loop-out recombination, (ii) clarification of the effects of a deficiency of recombination-related genes on the generation of large chromosomal deletions via loop-out recombination, (iii) clarification of the effect of chromosomal position on the frequency of chromosomal deletions by loop-out recombination, and (iv) development of techniques to facilitate large chromosomal deletions via loop-out recombination by introducing I-SceI-mediated DSBs into the target chromosome.
MATERIALS AND METHODS
Fungal strains, culture, media, and transformation.A list of the strains used in this study is presented in Table 1. Polypeptone-dextrin (PD) medium containing 1% polypeptone, 2% dextrin, 0.5% KH2PO4, 0.1% NaNO3, 0.05% MgSO4, and 0.1% Casamino Acids (pH 6.0) was used for liquid cultivation of the Aspergillus strains. Czapek-Dox (CZ) minimal medium plates supplemented with 20 mM uridine and 5-fluoroorotic acid (5-FOA) (1.5 mg/ml; Sigma, St. Louis, MO) were used for the positive selection of pyrG-deficient strains. The Aspergillus strains were transformed using a protoplast PEG method (17). The detailed protocol of transformation was previously described (21). The transformants obtained on the regeneration plates (1.2 M sorbitol-CZ) were transferred to and selected on CZ plates for further analysis.
Strains used in this study
I-SceI endonuclease-mediated loop-out recombination was carried out as follows: 50, 10, and 0 U of I-SceI endonuclease (New England BioLabs) were mixed with 2 × 107 of protoplasts in a 0.1-ml volume; 0.02 ml of PEG solution was used for transformation and the mixture was kept on ice for 40 min. After the addition of 0.1 ml of PEG solution, the mixture was spread on 1.2 M sorbitol-CZ plates containing 5-FOA (1.5 mg/ml) and regenerated. Tannic acid agar plates (1% glucose, 1% tannic acid, 0.2% NH4H2PO4, 0.2% KH2PO4, 0.1% MgSO4, and 1.5% agar; pH 7.5) were used for the detection of tannase activity. Malt medium (2% malt extract, 2% glucose, 0.1% polypeptone, and 10 mM uridine) was used as a complete medium. Information about the A. oryzae genome was obtained from the database of genomes analyzed at NITE (http://www.bio.nite.go.jp/dogan/Top).
To describe the names of genes concisely, we represented the gene number of chromosome 3 by using the prefix C instead of AO0900260000, e.g., C19 instead of AO090026000019, and represented the gene number of chromosome 8 by using the prefix H instead of AO0901030000, e.g., H74 instead of AO090026000074.
DNA techniques, PCR method, Southern hybridization, and array comparative genome hybridization.Genomic DNA of the Aspergillus strains was extracted as described previously (18). PCR amplification was carried out in a GeneAmp 9600 system (Applied Biosystems). For TA cloning, PCR fragments were amplified using ExTaq polymerase (Takara, Japan). Other PCR amplifications were carried out using KOD polymerase (Toyobo, Japan). Southern hybridizations were performed as previously described (21). Digoxigenin (DIG)-labeled probes were constructed using a DIG-PCR labeling kit (Roche Diagnostics, Germany). The probes were hybridized and detected using a DIG system as per the manufacturer's instructions (Roche Diagnostics). The oligonucleotide primers used in this study are listed in Table 2. The oligonucleotide array was purchased from Agilent Technology (Agilent). Details of the array were described in a previous study (9, 18).
Oligonucleotide primers used in this study
Sequencing of the pyrG open reading frame (ORF) at the pksA site of the Δrad52-ku70-5-FOA-5 and Δrad54-ku70-5-FOA-2 strains was carried out as follows. Three-kilobase fragments including pyrG were amplified using primers 7C3UM and pkdeLM from genomic DNA of the 5-FOA strains. The amplified fragment was purified and subjected to sequencing using primers P1097U, P1359L, and P1592L.
Construction of vectors for disruption of rad52, rad54, and rdh54 of A. oryzae.Vectors for the disruption of rad52, rad54, and rdh54 were constructed as follows. A 4.2-kb fragment containing rad52, amplified from A. oryzae genomic DNA by using primers rad52-448U and rad52-4636L, was cloned using the TOPO TA cloning kit (Invitrogen) to obtain pTrad52. Thereafter, pTrad52 was digested with PmaCI and SnaBI. A 6.9-kb fragment of PmaCI-SnaBI was dephosphorylated and ligated to a phosphorylated 2-kb fragment containing the pyrithiamine resistance gene (ptrA); this gene was amplified from pPTRI, a genome integration-type vector containing ptrA (Takara, Japan), by using primers ptrBg2482U and ptrBg4443L to obtain the rad52 disruption vector pRad52ptr1. A 6-kb fragment containing rad54, amplified from A. oryzae genomic DNA by using primers rad54-429U and rad54-6395L, was cloned using the TOPO TA cloning kit to obtain pTrad54. The pTrad54 was digested with HincII and dephosphorylated. The 8.1-kb fragment was ligated to a phosphorylated 2-kb fragment containing ptrA, amplified from pPTRI by using primers ptrBg2482U and ptrBg4443L, to obtain rad54 disruption vector pRad54ptr1. A 5.5-kb fragment containing rdh54 amplified from A. oryzae genomic DNA by using primers rdh54-599U and rdh54-6052L was cloned using the TOPO TA cloning kit to obtain pTrdh54. pTrdh54 was digested with PmaCI and SmaI. A dephosphorylated 7.2-kb fragment was ligated to a phosphorylated 2-kb fragment containing ptrA, amplified from pPRTI by using primers ptrBg2482U and ptrBg4443L, to obtain rdh54 disruption vector pRdh54ptr2.
Construction of disruption strains of the recombination-related genes.To analyze the disruption of nonhomologous end joining-related genes by loop-out recombination, Δku70, ΔligD, and ΔpyrG strains of A. oryzae (24) were transformed with p7C-3, a vector for a 100-kb deletion via loop-out recombination including a homologue of the aflatoxin (AF) cluster (23) (Fig. 1C). A Δku70 strain bearing p7C-3 (Δku70-7C3-1 strain), a ΔligD strain bearing p7C-3 (ΔligD-7C3-2 strain), and a ΔpyrG strain bearing p7C-3 (ΔpyrG-7C3-1 strain) were obtained. The correct integration of p7C-3 at the pksA site in the Δku70-7C3-1 and ΔligD-7C3-2 strains was confirmed by Southern hybridization (see Fig. S1 in the supplemental material). To avoid the difficulties of vector integration, the homologous recombination-related genes rad52, rad54, and rdh54 were disrupted using strains already bearing p7C-3. Further, the Δku70-7C3-1 strain (Δku70 strain of A. oryzae bearing p7C-3) was transformed with pRad52ptr1, pRad54ptr1, and pRdh54ptr2 (the vectors for Δrad52, rad54, and rdh54, respectively). Thus, Δku70-rad52, Δku70-rad54, and Δku70-rdh54 strains bearing p7C-3 were obtained. Confirmation of the disruption of rad52, rad54, and rdh54 in the Δku70-rad52, Δku70-rad54, Δku70-rdh54 strains is described in Fig. S2 to S4. The strains were designated as the Δrad52-ku70-7C3-1, Δrad54-ku70-7C3-1, and Δrdh54-ku70-7C3-1 strains. The disruption strains of recombination-related genes were used for further analysis.
Construction of vectors for deletion of a near-centromeric region in chromosome 8 by loop-out recombination.Vectors for deletion of a near-centromeric region, including the tannase gene, by loop-out recombination were constructed using a fusion-PCR technique and the Infusion cloning kit (Clontech) as follows. A 1.2-kb fragment amplified from A. oryzae genomic DNA using the primer pair TD1310LTU-TD2512LTL, a 1.1-kb fragment amplified using the primer pair TD2546RTU-TD3669RTL, and a 2.6-kb pyrG fragment amplified using the primer pair npyr219U-npyr2849L were purified and mixed. A 5-kb fragment amplified from the mixture using the primer pair TD1375U2-TD3646L2 was cloned using the TOPO TA cloning kit (Invitrogen) to obtain pTD2-1.
Fragments of 2 kb amplified from A. oryzae genomic DNA by using primer pairs 217kU-219kL, 271kU-273kL, 310kU-312kL, and 414kU-416kL were cloned into StuI-digested pTD2-1 by using the Infusion cloning kit (Clontech) to obtain vectors for 3-kb, 57-kb, 100-kb, and 200-kb deletions by loop-out recombination; the vectors were designated as pTD-IF-3k, pTD-IF-57k, pTD-IF100k, and pTD-IF-200k, respectively. To insert the 18-nucleotide (nt) I-SceI recognition sequence (5′-TAGGGATAACAGGGTAAT-3′) into pTD-IF100k and pTD-IF-200k, a 9-kb fragment amplified from pTD-IF100k using the primers 312kL-SceI and SceI-StuI, and a 9-kb fragment amplified from pTD-IF200k using the primers 418kL-SceI and SceI-StuI, were phosphorylated and ligated to obtain the vectors pTD-IF100k-SceI and pTD-IF200k-SceI, respectively. A scheme of the strategy used to construct the vectors is represented in Fig. S5 in the supplemental material.
RESULTS
Effect of a deficiency of recombination-related genes on loop-out recombination in a near-telomeric region.To assess the effect of a deficiency of recombination-related genes on the generation of chromosomal deletions by loop-out recombination, we used disruption strains of recombination-related genes. As a deletion target, a homologue of the aflatoxin (AF) cluster (Fig. 1C) was chosen. The homologue of the AF cluster of A. oryzae RIB40 strain is located in the near-telomeric region of chromosome 3; the range is approximately 12 to 82 kb from the telomere end. The strains bearing the vector for a 100-kb deletion, including the homologue of the AF cluster, were used as parental and positive control strains (Fig. 1C). An accurate 100-kb deletion of the targeted region is expected to occur during the normal loop-out deletion by homologous recombination. To clarify the genes involved in the generation of large chromosomal deletions via loop-out recombination in the near-telomeric region, we constructed disruption strains of ku70, ligD, rad52, rad54, and rdh54, the genes known to play crucial roles in the process of homologous recombination or nonhomologous end joining (NHEJ). The effects of the disruption of these genes on the generation of chromosomal deletion via loop-out recombination were elucidated by using Δku70, ΔligD, Δrad52-Δku70, Δrad54-Δku70, and Δrdh54-Δku70 strains of A. oryzae. From the conidiospores of each of the disruption strains, five 5-FOA-resistant colonies were obtained and analyzed by Southern hybridization and one 5-FOA-resistant colony was subjected to array comparative genome hybridization (aCGH) analysis. The result of aCGH is shown in Fig. 2. The vertical bar in Fig. 2 shows the ratio of the signal intensity of the hybridization probe of the deletion strain versus that of the control strain for each gene position. The 5-FOA-resistant strain from the Δku70 strain (namely, the Δku70-7C3-1 strain) had a precise 100-kb deletion of the target region generated by the homologous recombination (Fig. 2) (Δku70-5-FOA-1 strain). On the other hand, unintended chromosomal deletions were observed in the 5-FOA-resistant strains obtained from the ΔligD, Δrad52-ku70, and Δrad54-ku70 strains (Fig. 2) (ΔligD-5-FOA-2, Δrad52-ku70-5-FOA-2, and Δrad54-ku70-5-FOA-4 strains). The 5-FOA-resistant strain obtained from the ΔligD strain (ΔligD-7C3-2 strain) showed an extension of the deletion region, namely, the deletion of a 370-kb chromosomal region that included the end of chromosome 3 (Fig. 2) (ΔligD-5-FOA-2). In the case of the 5-FOA-resistant strains obtained from the Δrad52-ku70 strain (Δrad52-ku70-7C3-1 strain), the deletion occurred from the middle of the deletion target to the telomeric region, namely, the deletion of a 59-kb chromosomal region that included the end of chromosome 3 (Fig. 2) (Δrad52-ku70-5-FOA-2 strain). The 5-FOA-resistant strains obtained from the Δrad54-ku70 strain (Δrad54-ku70-7C3-1 strain) also showed an extensive deletion, namely, the deletion of a 150-kb chromosomal region that included the end of chromosome 3 (Fig. 2) (Δrad54-ku70-5-FOA-4 strain). The microarray used in this study was originally designed as an expression microarray, and most of the probes on the array were positioned only in open reading frames (ORFs). Therefore, accurate positions of the start and the end sites of the deletion could not be determined by the data from aCGH. However, the approximate positions of the deletions were determined. Detailed results of Southern hybridization are shown in Fig. S6A to S8C in the supplemental material. The results obtained from aCGH and Southern hybridization are summarized in Fig. 3. Precise loop-out recombinations were observed in all of the Δku70-5-FOA-resistant colonies and Δrdh54-ku70-5-FOA-resistant colonies, and the telomeric ends were retained in the 5-FOA-resistant colonies. On the other hand, in ΔligD, Δrad52-ku70, and Δrad54-ku70 strains, most of the 5-FOA-resistant colonies (5 of 5 in ΔligD, 4 of 5 in Δrad52-ku70, and 4 of 5 in Δrad54-ku70 strains) lost the telomeric region and showed extensive chromosomal deletions by illegitimate recombination (Fig. 3). The range of deletion in the Δrad52 and Δrad54 strains was narrower than that in the ΔligD strain. The rest of the 5-FOA colonies from the Δrad52-ku70 and Δrad54-ku70 strains did not contain a deletion (Fig. 3) (Δrad52-ku70-5-FOA-5 and Δrad54-ku70-5-FOA-2 strains). Fragments including ORFs of pyrG at the pksA region of the Δrad52-ku70-5-FOA-5 and Δrad54-ku70-5-FOA-2 strains were amplified by PCR and sequenced. A mutation causing exchange of serine at position 204 to pretermination in the ORF of pyrG was found in the Δrad52-ku70-5-FOA-5 strain. However, no mutation causing a deficiency of pyrG was found in the ORF of pyrG of the Δrad54-ku70-5-FOA-2 strain. Mutation in the Δrad54-ku70-5-FOA-2 strain might be sited in the promoter region of pyrG. These results suggest that the ligD, rad52, and rad54 genes play crucial roles in the precise loop-out recombination of a large chromosomal deletion in the near-telomeric region and that the ku70 and rdh54 genes have no apparent effect on precise loop-out recombination.
The effect of a deficiency of recombination-related genes on the generation of a large chromosomal deletion by loop-out recombination. The results of array comparative genome hybridization (aCGH) (deletion of the near-telomeric region in chromosome 3) using the Δku70, ΔligD, Δrad52-ku70, Δrad54-ku70, and Δrdh54-ku70 strains of A. oryzae are shown (boxes 1 to 5, respectively). The vertical bars in the figure indicate the log2 ratio of the signal intensity of the probes of each strain versus that of the control strain. The signals on the left side of the figure represent the telomere side, and those on the right side represent the centromere side.
Effect of a deficiency of recombination-related genes on the generation of a large chromosomal deletion by loop-out recombination. The results of loop-out recombination in each strain are summarized schematically. The top of the figure shows the position on chromosome 3. The gene number of chromosome 3 is represented by using prefix C instead of AO0900260000, e.g., C19 instead of AO090026000019. A blank space between the thick lines in the lower part of the figure represents the corresponding position of deletion on the chromosome in each 5-FOA strain. The numbers under each gene represent the distance from the end of chromosome 3 to the position of each gene.
The frequencies of occurrence of 5-FOA-resistant colonies in deletion of the near-telomeric region by loop-out recombination were almost the same among Δku70, Δrad52-ku70, and Δrad54-ku70 strains (Table 3). However, in the ΔligD strain, the frequency of 5-FOA-resistant colonies was approximately 10 times lower than in the Δku70 strain.
Frequency of deletions of the near-telomeric region by loop-out recombination in the ku70, rad52, rad54, and ligD disruption strains
Deletion of the near-centromeric region, including the tannase gene, by loop-out recombination.Next, to elucidate the position effect of the target chromosomal region on the generation of large deletions by loop-out recombination, we examined the frequency of chromosomal deletion by loop-out recombination in the near-centromeric region by using Δku70 and ΔligD strains. As a deletion target, a near-centromeric region of chromosome 8, including the tannase gene (AO090103000074) that is located 215 kb from the centromere, was selected (Fig. 1D). Our previous results showed that the 470-kb region, including the tannase gene on chromosome 8, could be deleted by replacement-type recombination and that no apparent change in the growth phenotype was observed in the deletion strain (24). First, we performed an aCGH experiment and confirmed that the 470-kb region on chromosome 8 of the deletion strain (KT6-2) was precisely deleted (Fig. 4). The hybridization signal corresponding to the 470 kb (AO090103000074 to AO090103000259) was clearly reduced in the strain (Fig. 4). The Δku70 and ΔligD strains were transformed with the deletion vectors for generating 3-kb (pTD-IF-3k), 57-kb (pTD-IF-57k), 100-kb (pTD-IF-100k), and 200-kb (pTD-IF-200k) deletions in the near-centromeric region (Fig. 1D). Subsequently, strains with 3-kb and 57-kb deletions by loop-out recombination in the near-centromeric region were obtained from the Δku70 and ΔligD strains by selecting on 5-FOA-CZ plates. The frequency of appearance of the 5-FOA-resistant colonies in the case of the strain with 3-kb deletions was approximately 10 times higher than that in the case of the strain with 57-kb deletions for both the Δku70 and ΔligD strains (Table 4). Further, most of the 5-FOA-resistant colonies obtained from the strains showed the expected targeted deletions and lost tannase activity. However, in the case of 100-kb and 200-kb deletions, 5-FOA-resistant colonies were obtained with low frequency (less than 1 × 10−7) and colonies with the targeted deletion in chromosome 8 were not observed (data not shown). As described previously, the 470-kb chromosomal region including the tannase gene was deleted by replacement-type recombination without major phenotypic change (24). These results suggest that the deletion of a large chromosomal region by loop-out recombination was restricted in the near-centromeric region of chromosome 8, presumably by the chromosomal structure.
Confirmation of the deletion of the near-centromeric region, including the tannase gene, by replacement-type recombination. The top of the figure shows the position on chromosome 8. The bottom of the figure shows the results of array comparative genome hybridization (aCGH). The vertical bars show the log2 ratio of the signal intensity of the probes of the deletion strain versus that of the control strain. The signals on the left side of the figure represent the centromere side, and those on the right side represent the telomere side.
Frequency of deletions of the near-centromeric region by loop-out recombination in ku70 and ligD disruption strains
Chromosomal deletion by loop-out recombination is facilitated by the introduction of DSBs in the near-centromeric region.From the above results, we surmised that the chromosomal structure hampered the generation of a large chromosomal deletion by loop-out recombination, and we suspected that loop-out recombination would be facilitated by destruction of the chromosome structure in the near-centromeric region. To test this hypothesis, we attempted to construct a large chromosomal deletion by introducing I-SceI endonuclease-mediated DNA double strand breaks (DSBs) into the chromosome, thereby destroying the chromosome structure (Fig. 5A). I-SceI endonuclease is a homing endonuclease that recognizes an 18-bp sequence (3) and has been used to analyze the DSB repair mechanism in many organisms (1). BLAST analysis showed that the I-SceI recognition sequence did not exist in the chromosomes of the A. oryzae RIB40 strain. Therefore, a unique DSB can be generated in the targeted region of the A. oryzae chromosome by introducing the I-SceI enzyme and the I-SceI recognition sequence into the chromosome. To introduce the I-SceI recognition sequence into the near-centromeric region of chromosome 8, we transformed the Δku70 and ΔligD strains with vectors bearing the I-SceI recognition site for 100-kb and 200-kb deletions by loop-out recombination (pTD-IF100k-SceI and pTD-IF200k-SceI, respectively).
Introduction of a chromosomal deletion by I-SceI endonuclease-mediated loop-out recombination (A) Schematic representation of the promotion of loop-out recombination by the introduction of I-SceI endonuclease-mediated DSBs into the chromosome. (B) 5-FOA-resistant colonies on regeneration plates. Protoplasts treated with 50 U, 10 U, and 0 U of I-SceI regenerated on the left, center, and right plates, respectively. (C) Phenotypes of the strains grown on tannic acid plates. TDIF2KD2F, 200-kb deletion strain; control, wild-type strain.
I-SceI-mediated loop-out recombination was carried out using the Δku70 strain bearing pTD-IF200k-SceI (TDIF2KD2) and the I-SceI homing enzyme, by using a modified restriction enzyme-mediated integration (REMI) technique (26). Protoplasts of the strain were regenerated on 1.2 M sorbitol-CZ containing 5-FOA. When 10 U or 0 U (control) of I-SceI was added to the protoplast, no 5-FOA-resistant colonies were obtained (Fig. 5B, right, center). However, 5-FOA-resistant colonies were obtained from the protoplast after the addition of 50 U of I-SceI (Fig. 5B, left), suggesting that the addition of a sufficiently large amount of I-SceI is critical for generating 5-FOA-resistant colonies via this reaction. The phenotypes of the 5-FOA-resistant colonies were observed on tannase plates (Fig. 5C). The control strain showed a clear halo on tannic acid plates (Fig. 5C, right). However, the TDIF2KD2F strain (5-FOA-resistant strain obtained from TDIF2KD2, Δku70 strain bearing pTD-IF200k-SceI) (Table 1) did not produce a halo (Fig. 5C, left), indicating that the 5-FOA strain lost tannase activity because of a chromosomal deletion. The 5-FOA-resistant colonies were analyzed by Southern hybridization and aCGH. The results of aCGH indicated that precise targeted loop-out deletion occurred in the 5-FOA-resistant strains (Fig. 6A). The hybridization signals of AO090103000073 to AO090103000152 (200 kb) in the aCGH were clearly reduced in the TDIF2KD2F strain (5-FOA-resistant strain obtained from TDIF2KD2, Δku70 strain bearing pTD-IF200k-SceI (Table 1; Fig. 6A, top). Moreover, aCGH results for TDIF1KD1F (5-FOA-resistant strain obtained from TDIF1KD1 strain, Δku70 strain bearing pTD-IF100k-SceI) (Table 1) showed reduced signals of AO090103000073 to AO090103000115 (100 kb) (Fig. 6A, bottom). These results indicated that 100-kb and 200-kb deletions by loop-out recombination were precisely generated in the Δku70 strain (Fig. 6B). The deletion frequency of the near-centromeric region by loop-out recombination with I-SceI in the ku70 and ligD disruption strains is shown in Table 5. The results of 5-FOA frequency obtained from I-SceI-mediated loop-out recombination were similar among the strains. The 100-kb and 200-kb deletions were achieved only when I-SceI was added in the Δku70 strain. In the case of the ΔligD strain, the 100-kb deletion was achieved after the addition of I-SceI. However, the 200-kb deletion was not obtained. The results of the loop-out recombination in the near-centromeric region in Δku70 and ΔligD strains are summarized in Fig. 6B. These results indicate that loop-out recombination in the near-centromeric region was facilitated by the introduction of I-SceI-mediated DSBs into the homologous sequence region for the loop-out recombination of the chromosome.
Deletion of the near-centromeric region, which include the tannase gene, by loop-out recombination. (A) Deletion of 200 kb (box 1) and 100 kb (box 2) by I-SceI endonuclease-mediated loop-out recombination in the Δku70 strain was confirmed by aCGH. (Box 1) TDIF2KD2F strain. (Box 2) TDIF1KD1F strain. (A, top) The position on chromosome 8 is shown at the top. The vertical bars at the bottom indicate the log2 ratio of the signal intensity of the probes of each strain versus that of the control strain. The signals on the left side of the figure represent the centromere side, and those on the right side represent the telomere side. (B) Summarized results of the deletion of the near-centromeric region by loop-out recombination. (Top) Position on chromosome 8. (Bottom) Open circles indicate that deletion strains of corresponding regions were obtained. The crosses indicate that deletion strains of the corresponding regions were not obtained.
Frequency of deletion of the near-centromeric region by loop-out recombination with I-SceI endonuclease in the ku70 and ligD disruption strains
DISCUSSION
In this study, we generated chromosomal deletions via loop-out recombination by using Δku70, ΔligD, Δrad52-ku70, Δrad54-ku70, and Δrdh54-ku70 strains of A. oryzae to elucidate the genes involved in loop-out recombination. A precise chromosomal deletion in the near-telomeric region was achieved in the Δku70 and Δrdh54-ku70 strains. On the other hand, unintended chromosomal deletions occurred in the near-telomeric region of the deletion strains obtained from the ΔligD, Δrad52-ku70, and Δrad54-ku70 strains. These results indicate that ligD, rad52, and rad54 play important roles in precise and stable loop-out recombination in the near-telomeric region and show that NHEJ is involved in the loop-out recombination between distant chromosomal sites. In S. cerevisiae, the rate of recombination between direct repeated sequences was reported to be drastically reduced in the rad52 mutant and increased in the rad54 mutant (12). The recombination between direct repeated sequences is thought to be a type of loop-out recombination. However, in the present study, no apparent change in the occurrence of 5-FOA-resistant colonies was observed among Δku70, Δrad52-ku70, and Δrad54-ku70 strains (Table 3), suggesting that the function of rad52 or rad54 we have seen in recombination in Aspergillus strain is different from that of yeast. In fact, the distance between recombination sites was much larger in Aspergillus (>100 kb) than in yeast (a few kb). Moreover, no precise loop-out recombination events were observed in the 5-FOA-resistant colonies obtained from the Δrad52-ku70 and Δrad54-ku70 Aspergillus strains, suggesting that the conditions for loop-out recombination in these species are different. Although the detailed functions of rad52 and rad54 in recombination are unclear, data obtained in yeast (12) are consistent with our observation that rad52 and rad54 play a role in loop-out recombination.
In the case of the ΔligD strain, illegitimate loop-out recombination occurred in the near-telomeric region of chromosome 3, and precise loop-out deletions were achieved in the near-centromeric region of chromosome 8. However, the frequency of 5-FOA-resistant colonies in the ΔligD strain was lower than in the other strains for deletion of the near-telomeric and near-centromeric regions. These results indicate that ligD plays a role in the generation of chromosomal deletion by loop-out recombination and is required in precise loop-out recombination in the near-centromeric region. Loop-out deletions of a few kb were previously reported in ΔligD strains (14, 24). In addition, instability of the near-telomeric region and deletion of chromosomal ends have been often observed in studies concerning the genome sequence analysis of several organisms that have a linear-type chromosome (25). These results suggest that the role played by ligD in loop-out recombination is related to the maintenance of the stability of the chromosomal structure during recombination. Possible mechanisms of loop-out recombination of a large chromosomal region and the generation of unintended chromosomal deletions in ΔligD strains are shown in Fig. 7. We hypothesized that the occurrence of DSBs during loop-out recombination and the repair of the DSBs through NHEJ are important in the generation of large chromosomal deletions by precise loop-out recombination. The introduction of DSBs into chromosomes occurs spontaneously during loop-out recombination of large chromosomal regions and is necessary for loop-out recombination between distant chromosomal sites. Dynamic conformational changes of the chromosome are necessary to complete loop-out recombination with a large chromosomal deletion (Fig. 7A). Spontaneous DSBs more frequently occur in the near-telomeric region than in the near-centromeric region. As a result, the frequency of large chromosomal deletions by loop-out recombination in near-telomeric regions is much higher than in near-centromeric regions (Fig. 7B). In the ΔligD strain, DSBs cannot be repaired by NHEJ because of complete inhibition of this mechanism; therefore, unintended chromosomal deletions occur in the near-telomeric and near-centromeric regions of the ΔligD strain (Fig. 7C). Strains with unintended chromosomal deletions of the near-telomeric region were obtained because these strains are viable; however, strains with unintended chromosomal deletions of the near-centromeric region were not obtained because these strains are lethal.
Possible mechanism for the generation of unintended chromosomal deletion in the ΔligD strain during loop-out recombination. (A) Introduction of DSBs into the chromosome occurs during the generation of a large chromosomal deletion by loop-out recombination and is necessary for loop-out recombination between distant sites on the chromosome. (B) DSBs occur more frequently in the near-telomeric region than in the near-centromeric region. As a result, the frequency of large chromosomal deletions by loop-out recombination in the near-telomeric region is higher than in the near-centromeric region. (C) Unintended chromosomal deletions occur in the near-telomeric and near-centromeric regions of the ΔligD strain. Unintended chromosomal deletion strains of the near-telomeric region are obtained because these strains are viable; however, unintended chromosomal deletion strains of the near-centromeric region are not obtained because these strains are lethal. The light gray bars in A and C show the homologous regions for loop-out recombination. The slanting striped pattern in B shows the target region.
These hypotheses are consistent with the observation that the frequency of 5-FOA-resistant colonies by loop-out recombination is lower in the ΔligD strain than in the Δku70 strain for the near-telomeric (Table 3) and near-centromeric (Table 4) regions. The reduction of the frequency of 5-FOA-resistant colonies in the ΔligD strain is probably influenced by the absence of DSB repair by NHEJ during loop-out recombination. As a result of illegitimate chromosomal deletion, most of the 5-FOA-resistant colonies from the ΔligD strain would be lethal. In the case of the Δku70 strain, the ku independent NHEJ pathway can supplement DSB repair.
In the present study, 3-kb and 57-kb chromosomal deletions were achieved by simple 5-FOA-mediated loop-out recombination in the near-centromeric region of chromosome 8. However, 100-kb and 200-kb deletions of the same locus were not achieved without the addition of I-SceI endonuclease. These results indicate that the loop-out-type recombination associated with large chromosomal deletions is difficult to achieve near the centromere. This is also supported by the data showing that the frequency of 5-FOA-resistant colonies in strains with the 3-kb chromosomal deletion construct was much higher than that in strains with the 57-kb deletion construct (Table 3). Our previous results indicate that the 470-kb region in the near-centromeric region of chromosome 8 does not contain genes that are essential for cell survival (Fig. 4) (24). The current results suggest that loop-out recombination is more sensitive to chromosomal structure than replacement-type recombination. Similar results were obtained in our previous study, i.e., replacement-type recombination generated large chromosomal deletions of the AF gene cluster region more readily than loop-out recombination (23, 24).
In the present study, 100-kb and 200-kb chromosomal deletions were generated by loop-out recombination in the near-centromeric region using I-SceI endonuclease. The parental strains of the 100-kb and 200-kb deletion strains contained a unique, introduced I-SceI restriction site at the target near-centromere chromosomal region of chromosome 8, suggesting that the large chromosomal deletions by loop-out recombination in the near-centromeric region were facilitated by the digestion of the chromosome at the homologous sequence for loop-out recombination. The 100-kb deletion in the near-centromeric region was also achieved in the ΔligD strain by using I-SceI. Since the I-SceI-mediated DSB was in the homologous sequence for loop-out recombination in the near-centromeric region, the deficiency of NHEJ in the ΔligD strain might cause a relatively small effect on the recombination.
The facilitation of chromosomal deletion by I-SceI endonuclease has been already reported in Escherichia coli and yeast (8, 11). The results obtained in the current study show that the destruction of chromosome structure by I-SceI endonuclease is useful for the construction of a large chromosomal deletion in the near-centromere region.
In conclusion, in this study, we investigated the genes involved in large chromosomal deletion by loop-out recombination and showed that NHEJ is involved in this process. This is the first report that NHEJ is involved in loop-out recombination. We concluded that ligD, rad52, and rad54 play crucial roles in precise loop-out recombination in the near-telomeric region and that loop-out recombination is more sensitive to chromosomal structure than replacement recombination. Our results clarified that loop-out recombination associated with a large chromosomal deletion was inhibited in the near-centromeric region and that recombination was facilitated by destruction of the chromosome structure through the activity of the I-SceI endonuclease. The results obtained in this study yield clues for researching recombination mechanisms of genomic DNA. The techniques described in this paper are also useful for the breeding of industrial strains by using genome information and genetic engineering techniques.
ACKNOWLEDGMENTS
We thank Yukio Senou, Michiyo Utsushikawa, and Michiyo Nishida for technical assistance.
FOOTNOTES
- Received 18 August 2011.
- Accepted 20 January 2012.
- Accepted manuscript posted online 27 January 2012.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/EC.05208-11.
- Copyright © 2012, American Society for Microbiology. All Rights Reserved.
REFERENCES
