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Prot Cell    2010, Vol. 1 Issue (5) : 478-490    https://doi.org/10.1007/s13238-010-0058-2      PMID: 21203963
RESEARCH ARTICLE
Drosophila RecQ5 is required for efficient SSA repair and suppression of LOH in vivo
Yixu Chen1,2, Wen Dui1,2,3, Zhongsheng Yu1,2, Changqing Li1, Jun Ma1,3, Renjie Jiao1()
1. State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Beijing 100101, China; 2. Graduate School of the Chinese Academy of Sciences, Beijing 100080, China; 3. Divisions of Biomedical Informatics and Developmental Biology, Cincinnati Children’s Hospital Research Foundation, 3333 Burnet Avenue, Cincinnati, OH 45229, USA
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Abstract

RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination. However, the specific pathway(s) in which it is involved and the underlining mechanism(s) remain poorly understood. We took advantage of genetic tools in Drosophila to investigate how Drosophila RecQ5 (dRecQ5) functions in vivo in homologous recombination-mediated double strand break (DSB) repair. We generated null alleles of dRecQ5 using the targeted recombination technique. The mutant animals are homozygous viable, but with growth retardation during development. The mutants are sensitive to both exogenous DSB-inducing treatment, such as gamma-irradiation, and endogenously induced double strand breaks (DSBs) by I-Sce I endonuclease. In the absence of dRecQ5, single strand annealing (SSA) -mediated DSB repair is compromised with compensatory increases in either inter-homologous gene conversion, or non-homologous end joining (NHEJ) when inter-chromosomal homologous sequence is unavailable. Loss of function of dRecQ5 also leads to genome instability in loss of heterozygosity (LOH) assays. Together, our data demonstrate that dRecQ5 functions in SSA-mediated DSB repair to achieve its full efficiency and in suppression of LOH in Drosophila.
Keywords Drosophila RecQ5      double strand break repair      homologous recombination      nonhomologous end joining      single strand annealing      RecQ helicase     
Corresponding Author(s): Jiao Renjie,Email:rjiao@sun5.ibp.ac.cn   
Issue Date: 01 May 2010
 Cite this article:   
Yixu Chen,Wen Dui,Zhongsheng Yu, et al. Drosophila RecQ5 is required for efficient SSA repair and suppression of LOH in vivo[J]. Prot Cell, 2010, 1(5): 478-490.
 URL:  
https://academic.hep.com.cn/pac/EN/10.1007/s13238-010-0058-2
https://academic.hep.com.cn/pac/EN/Y2010/V1/I5/478
Fig.1  Generation of mutant (): strategy and molecular characterization.
(A) Schematic diagram of genomic locus and gene targeting strategy. A mutant (* indicates the mutation site at the start codon) and the marker gene are circularized from the transgenic genome by FLP recombinase and linearized by the yeast restriction endonuclease I-Sce I before ends-in recombination resulting in a duplication of the locus (one wild type and the other mutant). Upon I-Cre I cutting and repairing via homologous recombination, the mutant can be selected with the loss of marker gene. (B) Sequence comparison of and the wild type (WT) indicating that the expected ATG (underlined) mutation to GCG (yielding a Not I cutting site) and a frame shift of the coding sequence in . Three mRNA splice isoforms of (dRecQ5-PA, -PB and-PC) are shown above the sequence comparison. (C) PCR in combination with Not I digestion (see materials and methods for details) shows that the artificial Not I site in the gives two bands of the PCR products (lane 3 and 4) while the PCR products from wild type flies cannot be cut by Not I (lane 2). Lane 1 is DNA ladders. (D) Western blot showing that the 53 kDa short forms of dRecQ5-PA and dRecQ5-PC, and the 121 KD form of dRecQ5-PB become undetectable in (lane 2). Lane 1 is the wild type control. The 43 kDa actin band is loading control. Molecular weights are indicated on the left of the panel.
Fig.1  Generation of mutant (): strategy and molecular characterization.
(A) Schematic diagram of genomic locus and gene targeting strategy. A mutant (* indicates the mutation site at the start codon) and the marker gene are circularized from the transgenic genome by FLP recombinase and linearized by the yeast restriction endonuclease I-Sce I before ends-in recombination resulting in a duplication of the locus (one wild type and the other mutant). Upon I-Cre I cutting and repairing via homologous recombination, the mutant can be selected with the loss of marker gene. (B) Sequence comparison of and the wild type (WT) indicating that the expected ATG (underlined) mutation to GCG (yielding a Not I cutting site) and a frame shift of the coding sequence in . Three mRNA splice isoforms of (dRecQ5-PA, -PB and-PC) are shown above the sequence comparison. (C) PCR in combination with Not I digestion (see materials and methods for details) shows that the artificial Not I site in the gives two bands of the PCR products (lane 3 and 4) while the PCR products from wild type flies cannot be cut by Not I (lane 2). Lane 1 is DNA ladders. (D) Western blot showing that the 53 kDa short forms of dRecQ5-PA and dRecQ5-PC, and the 121 KD form of dRecQ5-PB become undetectable in (lane 2). Lane 1 is the wild type control. The 43 kDa actin band is loading control. Molecular weights are indicated on the left of the panel.
Fig.2  Cellular and growth phenotypes of homozygous mutants.
(A) Comparison of wild type and animals at indicated time points of larval stages. WT indicates wild type larvae and M indicates mutant larvae. Scale bar, 600 μM. (B) Fractions of feeding larvae, wandering larvae and pupae at 114 h after egg deposition (AED) for both and wild type. (C) Fractions of pupae and adults at 212 h AED for both and wild type. (D) Loss-of-function of leads to spontaneous mitotic defects with increased frequency of aberrant chromosomes in early embryos (embryos that contain less than 5 mitotic abnormal chromosomal clusters (abnormal chr.) increased from ～2% to ～9%, while embryos that contain 5 and more mitotic abnormal chromosomal clusters increased from 0 to ～6%). Wild type and mutant embryos (0-2 h) were collected, fixed and stained for DNA (Topro3: blue) and phosphorylated histone H3 (PH3: red). Abnormal wild-type (above) and mutant (below) embryos at cycle 13 (prophase/metaphase) are shown on the left. Arrows indicate nuclei with mitotic defects. Scale bar, 50 μM. Statistic analysis is shown on the right. <0.01, Student’s t-test. More than 200 embryos were scored for each category.
Fig.2  Cellular and growth phenotypes of homozygous mutants.
(A) Comparison of wild type and animals at indicated time points of larval stages. WT indicates wild type larvae and M indicates mutant larvae. Scale bar, 600 μM. (B) Fractions of feeding larvae, wandering larvae and pupae at 114 h after egg deposition (AED) for both and wild type. (C) Fractions of pupae and adults at 212 h AED for both and wild type. (D) Loss-of-function of leads to spontaneous mitotic defects with increased frequency of aberrant chromosomes in early embryos (embryos that contain less than 5 mitotic abnormal chromosomal clusters (abnormal chr.) increased from ～2% to ～9%, while embryos that contain 5 and more mitotic abnormal chromosomal clusters increased from 0 to ～6%). Wild type and mutant embryos (0-2 h) were collected, fixed and stained for DNA (Topro3: blue) and phosphorylated histone H3 (PH3: red). Abnormal wild-type (above) and mutant (below) embryos at cycle 13 (prophase/metaphase) are shown on the left. Arrows indicate nuclei with mitotic defects. Scale bar, 50 μM. Statistic analysis is shown on the right. <0.01, Student’s t-test. More than 200 embryos were scored for each category.
Fig.3  mutants are sensitive to agents that cause DSBs.
(A) The third instar larvae of wild type and were treated with gamma irradiation at the indicated doses and allowed to recover and further develop at normal culture conditions. The mutants show significantly lower survival rate than the wild type control. (B) The survival rate is significantly reduced for the than the wild type flies upon the endogenous DSBs induction by I-Sce I endonuclease cutting at the transgene (; ). females were mated to males. flies in the offspring indicate DSB-occurring fraction while flies were scored as the endogenous control. More than 5000 flies in total were scored. Data were analyzed by Student’s t test and presented as mean±SEM with * for <0.05, ** for <0.01, and *** for <0.001.
Fig.3  mutants are sensitive to agents that cause DSBs.
(A) The third instar larvae of wild type and were treated with gamma irradiation at the indicated doses and allowed to recover and further develop at normal culture conditions. The mutants show significantly lower survival rate than the wild type control. (B) The survival rate is significantly reduced for the than the wild type flies upon the endogenous DSBs induction by I-Sce I endonuclease cutting at the transgene (; ). females were mated to males. flies in the offspring indicate DSB-occurring fraction while flies were scored as the endogenous control. More than 5000 flies in total were scored. Data were analyzed by Student’s t test and presented as mean±SEM with * for <0.05, ** for <0.01, and *** for <0.001.
Fig.4  mutants show different sensitivity to DSBs generated in different assays.
(A and B) Schematic diagrams (not to the scale) of the reconstitution assays and () for gap repair. Both and trangenes contain an incomplete yellow gene containing only the 5’ and 3’ segments that are separated by an I-Sce I cutting site. F1-5 is located at 7E7 on the X chromosome while F-R3 is located at 60F5 on the second chromosome. (C) Reconstituted somatic clones (arrows) in both females (left) and males (right). (D) Males are more sensitive to I-Sce I endonuclease than females in the absence of ( assay). (E) Females and males show similar sensitivities to I-Sce I induced DSBs regardless of the presence of in assay. females were mated to males in the presence or absence of . flies represent the DSB-occurring fraction, and the flies are used as an endogenous control. More than 5000 flies in total were scored. Data were analyzed by Student’s t test and presented as mean±SEM with * for <0.05, ** for <0.01, and *** for <0.001.
Fig.4  mutants show different sensitivity to DSBs generated in different assays.
(A and B) Schematic diagrams (not to the scale) of the reconstitution assays and () for gap repair. Both and trangenes contain an incomplete yellow gene containing only the 5’ and 3’ segments that are separated by an I-Sce I cutting site. F1-5 is located at 7E7 on the X chromosome while F-R3 is located at 60F5 on the second chromosome. (C) Reconstituted somatic clones (arrows) in both females (left) and males (right). (D) Males are more sensitive to I-Sce I endonuclease than females in the absence of ( assay). (E) Females and males show similar sensitivities to I-Sce I induced DSBs regardless of the presence of in assay. females were mated to males in the presence or absence of . flies represent the DSB-occurring fraction, and the flies are used as an endogenous control. More than 5000 flies in total were scored. Data were analyzed by Student’s t test and presented as mean±SEM with * for <0.05, ** for <0.01, and *** for <0.001.
Fig.5  mutation impairs SSA mediated DSB repair.
(A) Schematic illustration of the hemizygous assay. SSA and NHEJ frequencies in WT and mutant backgrounds are shown in B. (C) Schematic illustration of the homozygous assay. The insertion contains a part of the 3’ portion of (to the left of the I-Sce I site) and a copy of the functional . G0 generation that carries and UIE exhibits mosaic eyes. Among the G1 offspring, three kinds of eyes represent the results of the indicated repair pathways. × represents a mutated I-Sce I site due to imperfect NHEJ. (D) SSA, inter-homolog GC and NHEJ frequencies in WT and mutant background are shown. More than 200 flies were scored for each genotype. Data were analyzed by Student’s t test and presented as mean±SEM with * for <0.05, ** for <0.01, and *** for <0.001.
Fig.5  mutation impairs SSA mediated DSB repair.
(A) Schematic illustration of the hemizygous assay. SSA and NHEJ frequencies in WT and mutant backgrounds are shown in B. (C) Schematic illustration of the homozygous assay. The insertion contains a part of the 3’ portion of (to the left of the I-Sce I site) and a copy of the functional . G0 generation that carries and UIE exhibits mosaic eyes. Among the G1 offspring, three kinds of eyes represent the results of the indicated repair pathways. × represents a mutated I-Sce I site due to imperfect NHEJ. (D) SSA, inter-homolog GC and NHEJ frequencies in WT and mutant background are shown. More than 200 flies were scored for each genotype. Data were analyzed by Student’s t test and presented as mean±SEM with * for <0.05, ** for <0.01, and *** for <0.001.
Fig.6  mutants exhibit increased loss of heterozygosity (LOH).
(A) The genetic basis of the LOH assay with as a marker. In the heterozygous animals, when the wild type copy of is lost during the proliferation of wing cell precursors, the resulting clone will exhibit the recessive phenotype (multiple-wing hair). (B) mutation leads to an increase in the number of mutant clones (arrows) resulting from homologous recombination. (a) Individual wing hairs in an animal heterozygous for . Each hair follicle has one hair. (b) In an animal homozygous for , each hair follicle has two or more hairs. (c and c’) In an animal heterozygous for and homozygous for mutation, individual cells that lost the wild-type gene due to homologous recombination show two or more hairs (arrows). (C) The frequency of clones per wing in animals are significantly increased than in wild type. Nine wings for control and seven wings for mutants were scored for clones. Data were analyzed by Student’s t test and presented as mean±SEM with * for <0.05, ** for <0.01, and *** for <0.001. (D) The genetic basis of the LOH assay with as a marker (above). The numbers show the frequency of progenies with mosaic eyes. In wild type background, no flies with mosaic eyes were observed while in background mosaic flies appeared in all tubes tested.
Fig.6  mutants exhibit increased loss of heterozygosity (LOH).
(A) The genetic basis of the LOH assay with as a marker. In the heterozygous animals, when the wild type copy of is lost during the proliferation of wing cell precursors, the resulting clone will exhibit the recessive phenotype (multiple-wing hair). (B) mutation leads to an increase in the number of mutant clones (arrows) resulting from homologous recombination. (a) Individual wing hairs in an animal heterozygous for . Each hair follicle has one hair. (b) In an animal homozygous for , each hair follicle has two or more hairs. (c and c’) In an animal heterozygous for and homozygous for mutation, individual cells that lost the wild-type gene due to homologous recombination show two or more hairs (arrows). (C) The frequency of clones per wing in animals are significantly increased than in wild type. Nine wings for control and seven wings for mutants were scored for clones. Data were analyzed by Student’s t test and presented as mean±SEM with * for <0.05, ** for <0.01, and *** for <0.001. (D) The genetic basis of the LOH assay with as a marker (above). The numbers show the frequency of progenies with mosaic eyes. In wild type background, no flies with mosaic eyes were observed while in background mosaic flies appeared in all tubes tested.
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