Target DNA 염기서열 내에 존재하는 비상동성 간격이 상동성재조합을 이용한 클로닝 빈도에 미치는 영향

김재우,도은주,윤세련,정윤희,윤영호,임선희,선우양일,박인호,Kim Jae-Woo,Do Eun-Ju,Yoon Se-Lyun,Jeong Yun-Hee,Yoon Young-Ho,Leem Sun-Hee,Sunwoo Yangil,Park In-Ho 한국미생물학회 2005 미생물학회지 Vol.41 No.4

Transformation-associated recombination (TAR) 클로닝 법은 복잡한 게놈으로부터 염색체 내의 특정부위나 유전자를 선택적으로 분리할 수 있다. 이 방법은 목적 유전자에 근접한 작은 게놈DNA 염기서열 정보를 필요로 한다. 이 기술은 효모의 spheroplast transformation을 시키는 동안 목적으로 하는 유전자의 5' 또는 3' 서열을 포함하고 있는 TAR vector와 게놈DNA사이에서 일어나는 상동성재조합에 의해 이루어진다. 본 연구에서는 plasmid 모델시스템을 이용하여 target hooks 내에 존재하는 비상동성 염기서 열이 상동성재조합에 미치는 영향을 조사하였다. plasmid에 존재하는HIS3유전자와 변형시킨 his3-TRP1-his3 단편 사이의 상동성재조합의 효율은 $Ura^+$ 형질전환체의 형질분석에 의해 이루어졌다. $Ura^+$ 형질전환체의 수는 7종류의 서로 달리 변형된 his3-TRP1-his3 단편들을 사용하였을 매 거의 동일하게 나타났다. 그러나 $Trp^+His^+$ positive recombinants의 빈도는 변형된 his3-TRP1-his3 단편 내에 비상동성 영역에 부정확한 간격을 지닐 때 현저한 감소를 나타내었다. 이러한 결과로서, 부정확한 간격이 target hook과 substrate DNA 사이에 일어나는 상동성재조합을 방해하는 것으로 사료된다. 그러므로 이종간의 상동유전자를 클로닝 할 때에는 target hook내의 비상동성 염기서열이 존재한다면 이것이 정확한 간격을 지니는지 여부를 중요란 요인으로 고려해야 한다. Transformation-Associated Recombination (TAR) cloning technique allows selective isolation of chromosomal regions and genes from complex genomes. The procedure requires knowledge of relatively small genomic sequences that reside adjacent to the chromosomal region of interest. This technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector that has 5' and 3' gene targeting sequences. In this study, we examined the effect of non-homologous spacing sequence in target hooks on homologous recombination using a plasmid model system. The efficiency of homologous recombination between the modified his3-TRP1-his3 fragments and HlS3 gene on plasmid were analyzed by the characterization of $Ura^+$ transformants. The numbers of $Ura^+$ transformant showed same level when seven different modified his3-TRP1-his3 fragments were used. But the percentage of positive recombinants. $Trp^+His^-$, dramatically decreased when used the modified his3-TRP1-his3 fragments contained incorrect spacing of nonhomologous region. As a result, we suggest that incorrect spacing inhibits the homologous recombination between target hook and substrate DNA. Therefore, we should consider the correct spacing in target hook when the target hook are used for cloning of orthologue gene.

Meiotic prophase roles of Pds5 in recombination and chromosome condensation in budding yeast

Jeong Hwan Joo,Hyun Ah Kang,Keun Pil Kim,홍수길 한국미생물학회 2022 The journal of microbiology Vol.60 No.2

Genetic variation in eukaryotes is mediated during meiosis by the exchange of genetic material between homologous chromosomes to produce recombinant chromosomes. Cohesin is essential to promote proper chromosome segregation, chromosome morphogenesis, and recombination in meiotic cells. Cohesin consists of three main subunits–Smc1, Smc3, and the kleisin subunit Mcd1/Scc1 (Rec8 in meiosis)–and cohesin accessory factors. In Saccharomyces cerevisiae, the cohesin regulatory subunit Pds5 plays a role in homolog pairing, meiotic axis formation, and interhomolog recombination. In this study, we examine the prophase functions of Pds5 by performing physical analysis of recombination and three-dimensional high-resolution microscopy analysis to identify its roles in meiosis-specific recombination and chromosome morphogenesis. To investigate whether Pds5 plays a role in mitoticlike recombination, we inhibited Mek1 kinase activity, which resulted in switching to sister template bias by Rad51-dependent recombination. Reductions in double-strand breaks and crossover products and defective interhomolog recombination occurred in the absence of Pds5. Furthermore, recombination intermediates, including single-end invasion and double-Holliday junction, were reduced in the absence of Pds5 with Mek1 kinase inactivation compared to Mek1 kinase inactivation cells. Interestingly, the absence of Pds5 resulted in increasing numbers of chromosomes with hypercompaction of the chromosome axis. Thus, we suggest that Pds5 plays an essential role in recombination by suppressing the pairing of sister chromatids and abnormal compaction of the chromosome axis.

A simple analysis system for the estimation of recombination efficiency using fluorescence-activated cell sorting

Kim, M.S.,Kim, W.H.,Lee, G.M. Elsevier Science Publishers 2007 Journal of biotechnology Vol.127 No.3

A simple and accurate analysis system for the estimation of recombination efficiency in vivo using fluorescence-activated cell sorting (FACS) was designed and was subsequently used to compare the efficiency of recombination related to different spacer mutants. F<SUB>3</SUB> and F<SUB>5</SUB> mutant sequences were used for Flpe-mediated cassette exchange, and m2 and lox2272 mutant sequences were used for Cre-mediated cassette exchange due to their high incompatibility with wild-type sequences. The incompatibilities with wild-type were almost the same between mutant sequences. However, the recombination efficiencies were different. F<SUB>3</SUB> and m2 could mediate more efficient recombination than F<SUB>5</SUB> and lox2272, respectively. These results are consistent with the fact that the sequence of spacer region affects not only the reactivity upon wild-type sequence but also the recombination efficiency. It was also confirmed that the recombination process was mediated in a site-specific manner through PCR analysis using different sizes of exchange cassettes. In this experiment, the feasibility of the FACS analysis system for the estimation of recombination efficiency was verified. This system should be readily applicable for estimating recombination efficiency of other various mutant candidates, which will contribute to more precise and efficient site-specific recombination.

Recombination and optical properties of dislocations gliding at room temperature in GaN under applied stress

Vergeles, P.S.,Orlov, V.I.,Polyakov, A.Y.,Yakimov, E.B.,Kim, Taehwan,Lee, In-Hwan Elsevier 2019 Journal of Alloys and Compounds Vol.776 No.-

<P><B>Abstract</B></P> <P>The recombination and optical properties of dislocations in GaN introduced at room temperature by applied stress have been studied. It is observed that under the application of local shear stress of a few tens of MPa the dislocation glide in the parallel to the surface basal planes and in the planes intersecting the surface is activated at room temperature. It is shown that dislocations of dislocation half-loops gliding in the planes intersecting the surface can demonstrate both radiative and nonradiative recombination. Basal plane dislocations are shown to increase the nonradiative recombination rate. It is observed that the low-energy electron beam irradiation stimulates the dislocation glide both in the basal plane and the planes inclined to the surface, this effect being weaker for the basal plane. The analysis of electron irradiation effect on the dislocation related cathodoluminescence band suggests that this band is due to recombination involving complexes of point defects. These complexes are believed to be generated by gliding of the dislocation segments emerging at the surface.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Recombination properties of dislocations in GaN after a Vickers indentation are studied. </LI> <LI> Dislocation glide in the basal plane and planes inclined to the surface is activated at room temperatures. </LI> <LI> Dislocation half-loops gliding in the planes inclined to the surface demonstrate radiative recombination of charge carriers. </LI> <LI> Low-energy electron beam irradiation stimulates the dislocation glides. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Rad52/Rad59-dependent Recombination as a Means to Rectify Faulty Okazaki Fragment Processing

Lee, Miju,Lee, Chul-Hwan,Demin, Annie Albert,Munashingha, Palinda Ruvan,Amangyeld, Tamir,Kwon, Buki,Formosa, Tim,Seo, Yeon-Soo American Society for Biochemistry and Molecular Bi 2014 The Journal of biological chemistry Vol.289 No.21

<P>The correct removal of 5′-flap structures by Rad27 and Dna2 during Okazaki fragment maturation is crucial for the stable maintenance of genetic materials and cell viability. In this study, we identified <I>RAD52</I>, a key recombination protein, as a multicopy suppressor of <I>dna2-K1080E</I>, a lethal helicase-negative mutant allele of <I>DNA2</I> in yeasts. In contrast, the overexpression of Rad51, which works conjointly with Rad52 in canonical homologous recombination, failed to suppress the growth defect of the <I>dna2-K1080E</I> mutation, indicating that Rad52 plays a unique and distinct role in Okazaki fragment metabolism. We found that the recombination-defective Rad52-QDDD/AAAA mutant did not rescue <I>dna2-K1080E</I>, suggesting that Rad52-mediated recombination is important for suppression. The Rad52-mediated enzymatic stimulation of Dna2 or Rad27 is not a direct cause of suppression observed <I>in vivo</I>, as both Rad52 and Rad52-QDDD/AAAA proteins stimulated the endonuclease activities of both Dna2 and Rad27 to a similar extent. The recombination mediator activity of Rad52 was dispensable for the suppression, whereas both the DNA annealing activity and its ability to interact with Rad59 were essential. In addition, we found that several cohesion establishment factors, including Rsc2 and Elg1, were required for the Rad52-dependent suppression of <I>dna2-K1080E</I>. Our findings suggest a novel Rad52/Rad59-dependent, but Rad51-independent recombination pathway that could ultimately lead to the removal of faulty flaps in conjunction with cohesion establishment factors.</P>

Shu1 Promotes Homolog Bias of Meiotic Recombination in Saccharomyces cerevisiae

홍수길,김근필 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.5

Homologous recombination occurs closely between ho-mologous chromatids with highly ordered recombino-somes through RecA homologs and mediators. The present study demonstrates this relationship during the period of “partner choice” in yeast meiotic recombination. We have examined the formation of recombination intermediates in the absence or presence of Shu1, a member of the PCSS complex, which also includes Psy3, Csm2, and Shu2. DNA physical analysis indicates that Shu1 is essential for promoting the establishment of homolog bias during meiotic homologous recombination, and the partner choice is switched by Mek1 kinase activity. Furthermore, Shu1 pro-motes both crossover (CO) and non-crossover (NCO) pathways of meiotic recombination. The inactivation of Mek1 kinase allows for meiotic recombination to progress efficiently, but is lost in homolog bias where most double-strand breaks (DSBs) are repaired via stable intersister joint molecules. Moreover, the Srs2 helicase deletion cells in the budding yeast show slightly reduced COs and NCOs, and Shu1 promotes homolog bias inde-pendent of Srs2. Our findings reveal that Shu1 and Mek1 kinase activity have biochemically distinct roles in partner choice, which in turn enhances the understanding of the mechanism associated with the precondition for homolog bias.

Natural variation and dosage of the HEI10 meiotic E3 ligase control <i>Arabidopsis</i> crossover recombination

Ziolkowski, Piotr A.,Underwood, Charles J.,Lambing, Christophe,Martinez-Garcia, Marina,Lawrence, Emma J.,Ziolkowska, Liliana,Griffin, Catherine,Choi, Kyuha,Franklin, F. Chris H.,Martienssen, Robert A. Cold Spring Harbor Laboratory Press 2017 Genes & development Vol.31 No.3

<P>Here, Ziolkowski et al. combine high-throughput fluorescence methods to measure crossovers with natural <I>Arabidopsis</I> ecotypes in order to identify the first <I>trans</I>-acting modifier of meiotic recombination in plants. The authors found that HEI10, which encodes a conserved ubiquitin E3 ligase, naturally limits <I>Arabidopsis</I> crossovers and has the potential to influence the response to selection.</P><P>During meiosis, homologous chromosomes undergo crossover recombination, which creates genetic diversity and balances homolog segregation. Despite these critical functions, crossover frequency varies extensively within and between species. Although natural crossover recombination modifier loci have been detected in plants, causal genes have remained elusive. Using natural <I>Arabidopsis thaliana</I> accessions, we identified two major recombination quantitative trait loci (<I>rQTL</I>s) that explain 56.9% of crossover variation in Col×Ler F<SUB>2</SUB> populations. We mapped <I>rQTL1</I> to semidominant polymorphisms in <I>HEI10</I>, which encodes a conserved ubiquitin E3 ligase that regulates crossovers. Null <I>hei10</I> mutants are haploinsufficient, and, using genome-wide mapping and immunocytology, we show that transformation of additional <I>HEI10</I> copies is sufficient to more than double euchromatic crossovers. However, heterochromatic centromeres remained recombination-suppressed. The strongest <I>HEI10</I>-mediated crossover increases occur in subtelomeric euchromatin, which is reminiscent of sex differences in <I>Arabidopsis</I> recombination. Our work reveals that HEI10 naturally limits <I>Arabidopsis</I> crossovers and has the potential to influence the response to selection.</P>

N4SSB 단백질의 C-말단기의 7개의 아미노산이 N4SSB 단백질의 in vivo 활성에 미치는 영향

최미영,Choi, Mieyoung 한국미생물학회 1998 미생물학회지 Vol.34 No.4

Bacteriophage N4, a lytic phage specific for Esherichia coli K12 strain encodes single-stranded DNA-binding protein, N4SSB (bacteriophage N4-coded single-stranded DNA-binding protein). N4SSB protein is originally identified as a protein required for N4 DNA replication. N4SSB protein is also required for N4 late transcription, which is catalyzed by E. coli ${\sigma}^{70}$ RNA polymerase. N4 late transcription does not occur until N4SSB protein is synthesized. Recently it is reported that N4SSB protein is essential for N4 DNA recombination. Therefore N 4SSB protein is a multifunctional protein required for N4 DNA replication, late transcription, and N4 DNA recombination. In this study, a variety of mutant N4SSB proteins containing internal deletions or substitutions were constructed to define and characterize domains important for N4 DNA replication, late transcription, and N4 DNA recombination. Test for the ill vivo activity of these mutant N4SSBs for N4 DNA replication, late transcription, and N4 DNA recombination was examined. The results suggest that C-terminal 7 amino acid residues are important for the activity of N4SSB. Three lysine residues, which are contained in this region play important roles on N4SSB activity. Esherichia coli(E. coli) K12 균주를 숙주세포로 삼는 박테리오파아지인 N4는 single-stranded DNA에 결합하는 단백질인 N4SSB(bacteriophage N4-coded single-stranded DNA-binding protein) 단백질을 만든다. N4SSB 단백질은 N4 DNA replication 뿐만 아니라 late transcription과 N4 DNA recombination에도 필요한 여러 가지 기능을 가진 단백질이다. N4 late transcription은 숙주세포인 E. coli의 $E{\sigma}^{70}$ RNA polymerase에 의해서 수행이 되나 N4SSB 단백질을 반드시 필요로 하기 때문에 N4SSB 단백질이 생성될 때까지는 N4 late promoter로부터 RNA 합성이 일어나지 않는다. 본 연구에서는 N4SSB의 N4 DNA replication과 late transcription, 그리고 N4 DNA recombination에 필요한 영역(domain)을 알아내기 위해서 여러 가지 돌연변이형 N4SSB 단백질을 만들어 N4 DNA replication과 late transcription, 그리고 N4 DNA recombination의 3가지 작용에 대한 in vivo 활성을 조사 분석하였다. 그 결과 N4SSB 단백질의 C-말단기에 있는 7개의 아미노산이 N4SSB 단백질의 활성에 중요하다는 것을 알 수 있었다. 특히 C-말단기의 7개의 아미노산에는 세 개의 lysine이 포함되어 있는데 이 lysine이 N4SSB 단백질의 활성에 중요한 역할을 한다는 것이 제시되었다.

Effects of Recombination on the Pathogenicity and Evolution of Pepper mottle virus

Jonson, Miranda Gilda,Seo, Jang-Kyun,Cho, Hong-Soo,Kim, Jeong-Soo,Kim, Kook-Hyung The Korean Society of Plant Pathology 2009 Plant Pathology Journal Vol.25 No.4

The analysis of the full length genome of Korean isolates of Pepper mottle virus (PepMoV) in previous study showed molecular variations and are found to be related to symptom variation and pathogenicity (Kim et al., 2009, Virus Res. 144:83-88). To fully understand the molecular variation of PepMoV in Korea, we further assessed the role of RNA recombination to biological variation and evolution of PepMoV. Full-length genome of a total of 17 Korean-PepMoV and 2 American (CA and FL) isolates were examined for possible detection of genetic recombination using different recombination detections programs and detected 5 and 8 tentative recombination events using RDP3 and Splits Tree4 programs, respectively. Interestingly, tentative recombinants detected such as isolates 57, 134 and 217 were previously identified as severe isolates and 205135 and 205136 as differentiating isolates (Kim et al., 2009, Virus Res. 144:83-88). In addition, recombination was frequently detected in the Vb isolate, the first PepMoV isolate reported in Korea, suggesting significant involvement in the evolution of PepMoV in Korea. These initial results of our recombination analyses among PepMoV isolates in Korea may serve as clues to further investigate the biological variations and evolution of PepMoV brought about by recombination.

DNA-dependent Protein Kinase Mediates V(D)J Recombination via RAG2 Phosphorylation

Hah, Young-Sool,Lee, Jung-Hwa,Kim, Deok-Ryong Korean Society for Biochemistry and Molecular Biol 2007 Journal of biochemistry and molecular biology Vol.40 No.3

V(D)J recombination, a site-specific gene rearrangement process occurring during the lymphocyte development, begins with DNA double strand breaks by two recombination activating gene products (RAG1/2) and finishes with the repair process by several proteins including DNA-dependent protein kinase (DNA-PK). In this report, we found that RAG2 was specifically phosphorylated by DNA-PK at the $365^{th}$ serine residue, and this phosphorylated RAG2 affected the V(D)J recombination activity in cells in the GFP expression-based assay. While the V(D)J recombination activity between wild-type RAG2 and mutant S365A RAG2 in the assay using a signal joint substrate was undistinguishable in DNA-PK deficient cells (M059J), the activity with wild-type RAG2 was largely increased in DNA-PK proficient cells (M059K) in comparison with mutant RAG2, suggesting that RAG2 phosphorylation by DNA-PK plays a crucial role in the signal joint formation during V(D)J recombination.