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Stephen C Kowalczykowski - One of the best experts on this subject based on the ideXlab platform.
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a molecular throttle the Recombination Hotspot χ controls dna translocation by the recbcd helicase
Cell, 2003Co-Authors: Maria Spies, Piero R Bianco, Mark S Dillingham, Naofumi Handa, Ronald J. Baskin, Stephen C KowalczykowskiAbstract:Abstract RecBCD enzyme is a heterotrimeric helicase/nuclease that initiates homologous Recombination at double-stranded DNA breaks. Several of its activities are regulated by the DNA sequence χ (5′-GCTGGTGG-3′), which is recognized in cis by the translocating enzyme. When RecBCD enzyme encounters χ, the intensity and polarity of its nuclease activity are changed, and the enzyme gains the ability to load RecA protein onto the χ-containing, unwound single-stranded DNA. Here, we show that interaction with χ also affects translocation by RecBCD enzyme. By observing translocation of individual enzymes along single molecules of DNA, we could see RecBCD enzyme pause precisely at χ. Furthermore, and more unexpectedly, after pausing at χ, the enzyme continues translocating but at approximately one-half the initial rate. We propose that interaction with χ results in an enzyme in which one of the two motor subunits, likely the RecD motor, is uncoupled from the holoenzyme to produce the slower translocase.
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eLS - RecBCD helicase/nuclease
Encyclopedia of Life Sciences, 2001Co-Authors: Deana A Arnold, Stephen C KowalczykowskiAbstract:The RecBCD enzyme of Escherichia coli participates in several aspects of DNA Recombination and repair. It is essential to the main pathway of genetic homologous Recombination, where it contributes to the exchange of genetic material between homologous DNA molecules (i.e. conjugal Recombination), and to the Recombinational repair of potentially lethal chromosomal double-stranded breaks. Keywords: RecBCD; Recombination; Hotspot; DNA repair; E. coli
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recbcd helicase nuclease
eLS, 2001Co-Authors: Deana A Arnold, Stephen C KowalczykowskiAbstract:The RecBCD enzyme of Escherichia coli participates in several aspects of DNA Recombination and repair. It is essential to the main pathway of genetic homologous Recombination, where it contributes to the exchange of genetic material between homologous DNA molecules (i.e. conjugal Recombination), and to the Recombinational repair of potentially lethal chromosomal double-stranded breaks. Keywords: RecBCD; Recombination; Hotspot; DNA repair; E. coli
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a novel 11 nucleotide variant of χ χ one of a class of sequences defining the escherichia coli Recombination Hotspot χ
Journal of Molecular Biology, 2000Co-Authors: Deana A Arnold, Naofumi Handa, Ichizo Kobayashi, Stephen C KowalczykowskiAbstract:In wild-type Escherichia coli, recognition of the Recombination Hotspot, chi (5'-GCTGGTGG-3'), by the RecBCD enzyme is central to homologous Recombination. However, in the recC* class of RecBCD mutants, stimulation of Recombination by the canonical chi sequence is not detectable, but the levels of homologous Recombination are nearly wild-type. In vivo studies demonstrate that a member of this class of mutants, the recC1004 allele, encodes an enzyme that responds to a novel variant of chi, termed chi* (5'-GCTGGTGCTCG-3'). Here, we establish that, in vitro, the chi* sequence is recognized more efficiently by the RecBC(1004)D enzyme than is the wild-type chi. This is manifest by both a greater modification of nuclease activity and a higher stimulation of RecA protein-mediated joint molecule formation at chi* than at chi. Sequencing of the recC1004 gene revealed that it contains a frameshift mutation, which results in a replacement of nine of the wild-type amino acid residues by eight in the mutant protein, and defines a locus that is important for the specificity of chi-recognition. In addition, we show that this novel, 11 nucleotide chi* sequence also regulates the wild-type RecBCD enzyme, supporting the notion that variants of the canonical chi constitute a class of sequences that regulate the Recombination function of RecBCD enzyme.
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the Recombination Hotspot chi is recognized by the translocating recbcd enzyme as the single strand of dna containing the sequence 5 gctggtgg 3
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Piero R Bianco, Stephen C KowalczykowskiAbstract:The RecBCD enzyme of Escherichia coli func- tions in the seemingly disparate roles of homologous recom- bination and the degradation of DNA. Which of these two roles it assumes is regulated by the 8-base Recombination Hotspot, Chi. Using double-stranded DNA substrates that are hetero- duplex at the Chi locus we have established the determinants for Chi recognition. Our results show that an actively trans- locating RecBCD enzyme requires only the sequence infor- mation in the 5*-GCTGGTGG-3*-containing strand to recog- nize and to be regulated by Chi. Furthermore, the RecBCD enzyme can translocate through DNA heteroduplex bubbles as large as 22 bases, and still recognize a Chi sequence embedded in this region. This implies that recognition of Chi occurs following the unwinding of the DNA.
Gerald R Smith - One of the best experts on this subject based on the ideXlab platform.
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Optimizing the Nucleotide Sequence of a Meiotic Recombination Hotspot in Schizosaccharomyces pombe
Genetics, 2005Co-Authors: Walter W. Steiner, Gerald R SmithAbstract:The ade6-M26 mutation of Schizosaccharomyces pombe created a meiotic Recombination Hotspot. Previous analyses indicated that the heptamer 5′-ATGACGT-3′ was necessary and sufficient for Hotspot activity; the Atf1-Pcr1 transcription factor binds to this sequence and activates M26. After finding cases in which the M26 heptamer in ade6 was, surprisingly, not active as a Hotspot, we used an in vitro selection method (SELEX) that revealed an 18-bp consensus sequence for Atf1-Pcr1 binding, 5′-GNVTATGACGTCATNBNC-3′, containing the M26 heptamer at its core. Using this consensus sequence as a guide, we made mutations on each side of the heptamer at two separate sites in ade6. These mutations increased the intracellular Hotspot activity of the heptamer, in some cases by >15-fold. These results show that M26, the eukaryotic Recombination Hotspot with the most precisely defined nucleotide sequence, is larger than previously thought, and they provide valuable information for clarifying the role of M26, and perhaps other Hotspots, in meiotic Recombination.
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A 160-bp Palindrome Is a Rad50·Rad32-Dependent Mitotic Recombination Hotspot in Schizosaccharomyces pombe
Genetics, 2002Co-Authors: Joseph A. Farah, Kenichi Mizuno, Kunihiro Ohta, Edgar Hartsuiker, Gerald R SmithAbstract:Abstract Palindromic sequences can form hairpin and cruciform structures that pose a threat to genome integrity. We found that a 160-bp palindrome (an inverted repeat of 80 bp) conferred a mitotic Recombination Hotspot relative to a control nonpalindromic sequence when inserted into the ade6 gene of Schizosaccharomyces pombe. The Hotspot activity of the palindrome, but not the basal level of Recombination, was abolished by a rad50 deletion, by a rad50S “separation of function” mutation, or by a rad32-D25A mutation in the nuclease domain of the Rad32 protein, an Mre11 homolog. We propose that upon extrusion of the palindrome the Rad50·Rad32 nuclease complex recognizes and cleaves the secondary structure thus formed and generates a recombinogenic break in the DNA.
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A 160-bp palindrome is a Rad50.Rad32-dependent mitotic Recombination Hotspot in Schizosaccharomyces pombe
Genetics, 2002Co-Authors: Joseph A. Farah, Kenichi Mizuno, Kunihiro Ohta, Edgar Hartsuiker, Gerald R SmithAbstract:Palindromic sequences can form hairpin and cruciform structures that pose a threat to genome integrity. We found that a 160-bp palindrome (an inverted repeat of 80 bp) conferred a mitotic Recombination Hotspot relative to a control nonpalindromic sequence when inserted into the ade6 gene of Schizosaccharomyces pombe. The Hotspot activity of the palindrome, but not the basal level of Recombination, was abolished by a rad50 deletion, by a rad50S "separation of function" mutation, or by a rad32-D25A mutation in the nuclease domain of the Rad32 protein, an Mre11 homolog. We propose that upon extrusion of the palindrome the Rad50.Rad32 nuclease complex recognizes and cleaves the secondary structure thus formed and generates a recombinogenic break in the DNA.
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A Family of cAMP-Response-Element-Related DNA Sequences With Meiotic Recombination Hotspot Activity in Schizosaccharomyces pombe
Genetics, 2000Co-Authors: Mary E. Fox, Takatomi Yamada, Kunihiro Ohta, Gerald R SmithAbstract:The heptamer sequence ATGACGT is essential for activity of the M26 meiotic Recombination Hotspot in the ade6 gene of Schizosaccharomyces pombe. Hotspot activity is associated with binding of the heterodimeric transcription factor Atf1·Pcr1 to M26. We have found that the sequences (C/T/G) TGACGT also bound Atf1·Pcr1 and acted as meiotic Hotspots, but unlike M26 they must be followed by A or C for Atf1·Pcr1 binding and Hotspot activity. The basis of the Hotspot activity of CTGACGTA ( ade6-3013 ) appears to be identical to that of M26 : Hotspot activity of both sequences was abolished in cells mutant for atf1, pcr1, spc1 , or wis1 and was undetectable in mitotic Recombination and in meiotic Recombination when located on a plasmid. Both Hotspot sequences were sites of micrococcal nuclease hypersensitivity in meiotic chromatin, suggesting that they create an open chromatin structure during meiosis at the site of the Hotspots. The newly identified Hotspot sequences (C/T/G)TGACGT(A/C) and M26 are closely related to the cAMP response element (CRE) consensus sequence for binding of cAMP-responsive transcription factors such as Atf1·Pcr1, suggesting a link between transcription and meiotic Recombination. These results significantly expand the list of identified sequences with meiotic Recombination Hotspot activity in S. pombe from a single sequence to a family of CRE-related sequences.
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Active and inactive transplacement of the M26 Recombination Hotspot in Schizosaccharomyces pombe.
Genetics, 1995Co-Authors: J B Virgin, J Metzger, Gerald R SmithAbstract:Abstract The ade6-M26 mutation of the fission yeast Schizosaccharomyces pombe creates a meiotic Recombination Hotspot that elevates ade6 intragenic Recombination approximately 10-15-fold. A heptanucleotide sequence including the M26 point mutation is required but not sufficient for Hotspot activity. We studied the effects of plasmid and chromosomal context on M26 Hotspot activity. The M26 Hotspot was inactive on a multicopy plasmid containing M26 embedded within 3.0 or 5.9 kb of ade6 DNA. Random S. pombe genomic fragments totaling approximately 7 Mb did not activate the M26 Hotspot on a plasmid. M26 Hotspot activity was maintained when 3.0-, 4.4-, and 5.9-kb ade6-M26 DNA fragments, with various amounts of non-S. pombe plasmid DNA, were integrated at the ura4 chromosomal locus, but only in certain configurations relative to the ura4 gene and the cointegrated plasmid DNA. Several integrations created new M26-independent Recombination Hotspots. In all cases the non-ade6 DNA was located > 1 kb from the M26 site, and in some cases > 2 kb. Because the chromosomal context effect was transmitted over large distances, and did not appear to be mediated by a single discrete DNA sequence element, we infer that the local chromatin structure has a pronounced effect on M26 Hotspot activity.
Thomas D. Petes - One of the best experts on this subject based on the ideXlab platform.
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The Compact Chromatin Structure of a Ty Repeated Sequence Suppresses Recombination Hotspot Activity in Saccharomyces cerevisiae
Molecular cell, 2004Co-Authors: Shay Ben-aroya, Thomas D. Petes, Piotr A. Mieczkowski, Martin KupiecAbstract:Recombination between repeated DNA sequences can have drastic consequences on the integrity of the genome. Repeated sequences are abundant in most eukaryotes, yet the mechanism that prevents Recombination between them is currently unknown. Ty elements, the main family of dispersed repeats in Saccharomyces cerevisiae, exhibit low levels of exchange. Other regions in the genome have relatively high rates of meiotic Recombination (Hotspots). We show that a Ty element adjacent to the HIS4 Recombination Hotspot substantially reduces its activity, eliminating local DSB formation. We demonstrate that the Ty has a closed (nuclease-insensitive) chromatin configuration that is also imposed on the flanking DNA sequences. The compact chromatin structure is determined by sequences at the N terminus of the Ty. Increased binding of the Rap1 protein to the Hotspot restores both open chromatin conformation and DSB formation. The chromatin configuration of Ty elements precludes initiation of Recombination, thus preventing potentially lethal exchanges between repeated sequences.
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maximal stimulation of meiotic Recombination by a yeast transcription factor requires the transcription activation domain and a dna binding domain
Genetics, 1999Co-Authors: David T Kirkpatrick, Qingqing Fan, Thomas D. PetesAbstract:The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic Recombination Hotspot. Although the activity of this Hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of Recombination. We find that the Recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein. We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate Recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4. In addition, we find Recombination Hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors. We suggest that yeast cells have two types of Recombination Hotspots, α (transcription factor dependent) and β (transcription factor independent).
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fine structure mapping of meiosis specific double strand dna breaks at a Recombination Hotspot associated with an insertion of telomeric sequences upstream of the his4 locus in yeast
Genetics, 1996Co-Authors: Thomas D. PetesAbstract:Meiotic Recombination in Saccharomyces cerevisiae is initiated by double-strand DNA breaks (DSBs). Using two approaches, we mapped the position of DSBs associated with a Recombination Hotspot created by insertion of telomeric sequences into the region upstream of HIS4. We found that the breaks have no obvious sequence specificity and localize to a region of ~50 bp adjacent to the telomeric insertion. By mapping the breaks and by studies of the exonuclease III sensitivity of the broken ends, we conclude that most of the broken DNA molecules have blunt ends with 3'-hydroxyl groups.
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analysis of meiotic Recombination events near a Recombination Hotspot in the yeast saccharomyces cerevisiae
Current Genetics, 1994Co-Authors: Michael A White, Thomas D. PetesAbstract:The region of yeast chromosome III between the HIS4 and LEU2 genes has an unusually high frequency of meiotic Recombination. In order to determine the pattern of cross-over and gene conversion events, we constructed a strain with a number of heterozygous markers in this 25-kb interval. We found that very high levels of reombination are localized to regions of DNA near HIS4. In addition, analysis of the patterns of co-conversion of adjacent markers suggests that there is more than one initiation site contributing to Recombination of HIS4.
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transcription factors are required for the meiotic Recombination Hotspot at the his4 locus in saccharomyces cerevisiae
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Michael A White, Margaret Dominska, Thomas D. PetesAbstract:Abstract The full activity of a Recombination initiation site located 5' of HIS4 requires the binding of the transcription factors RAP1, BAS1, and BAS2. Two RAP1 binding sites can substitute for the wild-type initiation site. A 51-bp region of telomeric DNA inserted upstream of either HIS4 or ARG4 very strongly stimulates Recombination. We suggest that the ability of transcription factors to induce Recombination is a consequence of an altered chromatin structure that favors the entry of proteins that initiate Recombination, rather than an effect of these factors on transcription.
Mary Carrington - One of the best experts on this subject based on the ideXlab platform.
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molecular mapping of a Recombination Hotspot located in the second intron of the human tap2 locus
American Journal of Human Genetics, 1995Co-Authors: Michael Cullen, Henry A Erlich, William Klitz, Mary CarringtonAbstract:Recombination across the HLA class II region is not randomly distributed, as indicated by both strong linkage disequilibrium within the 100 kb encompassing the DRB1-DQA1-DQB1 loci and complete equilibrium between TAP1 and TAP2, the closest variant sites of which are <15 kb. In an attempt to explain these observations, 39 novel polymorphic markers in a region encompassing the TAP, LMP, and DOB genes were used to delineate the site of crossover in 11 class II recombinant chromosomes. SSCP demonstrated that two Recombination events occurred within an 850-bp interval in the second intron of TAP2, which separates the variant sites of TAP1 and TAP2. These data indicate the presence of a Recombination Hotspot, the first to be identified from the analysis of familial transmission in the human major histocompatibility complex. The region of crossover was cloned and sequenced from one of the recombinants, further defining the crossover site to a 138-bp segment nested within the 850-bp region. This represents the most precisely defined region of Recombination in the human genome. 44 refs., 3 figs., 2 tabs.
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Molecular mapping of a Recombination Hotspot located in the second intron of the human TAP2 locus.
American journal of human genetics, 1995Co-Authors: Michael Cullen, Henry A Erlich, William Klitz, Mary CarringtonAbstract:Recombination across the HLA class II region is not randomly distributed, as indicated by both strong linkage disequilibrium within the 100 kb encompassing the DRB1-DQA1-DQB1 loci and complete equilibrium between TAP1 and TAP2, the closest variant sites of which are
Kunihiro Ohta - One of the best experts on this subject based on the ideXlab platform.
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distinct chromatin modulators regulate the formation of accessible and repressive chromatin at the fission yeast Recombination Hotspot ade6 m26
Molecular Biology of the Cell, 2007Co-Authors: Kouji Hirota, Kenichi Mizuno, Takehiko Shibata, Kunihiro OhtaAbstract:Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) regulate transcription and Recombination via alteration of local chromatin configuration. The ade6-M26 allele of Schizosaccharomyces pombe creates a meiotic Recombination Hotspot that requires a cAMP-responsive element (CRE)-like sequence M26, the Atf1/Pcr1 heterodimeric ATF/CREB transcription factor, the Gcn5 HAT, and the Snf22 SWI2/SNF2 family ADCR. Chromatin alteration occurs meiotically around M26, leading to the activation of meiotic Recombination. We newly report the roles of other chromatin remodeling factors that function positively and negatively in chromatin alteration at M26: two CHD-1 family ADCRs (Hrp1 and Hrp3), a Spt-Ada-Gcn5 acetyltransferase component (Ada2), and a member of Moz-Ybf2/Sas3-Sas2-Tip60 family (Mst2). Ada2, Mst2, and Hrp3 are required for the full activation of chromatin changes around M26 and meiotic Recombination. Acetylation of histone H3 around M26 is remarkably reduced in gcn5Δ, ada2Δ and snf22Δ, suggesting cooperative functions of these HAT complexes and Snf22. Conversely, Hrp1, another CHD-1 family ADCR, maintains repressive chromatin configuration at ade6-M26. Interestingly, transcriptional initiation site is shifted to a site around M26 from the original initiation sites, in couple with the histone acetylation and meiotic chromatin alteration induced around 3′ region of M26, suggesting a collaboration between these chromatin modulators and the transcriptional machinery to form accessible chromatin. These HATs and ADCRs are also required for the regulation of transcription and chromatin structure around M26 in response to osmotic stress. Thus, we propose that multiple chromatin modulators regulate chromatin structure reversibly and participate in the regulation of both meiotic Recombination and stress-induced transcription around CRE-like sequences.
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roles of histone acetylation and chromatin remodeling factor in a meiotic Recombination Hotspot
The EMBO Journal, 2004Co-Authors: Takatomi Yamada, Kenichi Mizuno, Kouji Hirota, Ning Kon, Wayne P Wahls, Edgar Hartsuiker, Hiromu Murofushi, Takehiko Shibata, Kunihiro OhtaAbstract:Histone acetyltransferases (HATs) and ATP-dependent chromatin remodeling factors (ADCRs) are involved in selective gene regulation via modulation of local chromatin configuration. Activation of the Recombination Hotspot ade6-M26 of Schizosaccharomyces pombe is mediated by a cAMP responsive element (CRE)-like sequence, M26, and a heterodimeric ATF/CREB transcription factor, Atf1·Pcr1. Chromatin remodeling occurs meiotically around M26. We examined the roles of HATs and ADCRs in chromatin remodeling around M26. Histones H3 and H4 around M26 were hyperacetylated in an M26- and Atf1-dependent manner early in meiosis. SpGcn5, the S. pombe homolog of Gcn5p, was required for the majority of histone H3 acetylation around M26 in vivo. Deletion of gcn5+ caused a significant delay in chromatin remodeling but only partial reduction of M26 meiotic Recombination frequency. The snf22+ (a Swi2/Snf2-ADCR homologue) deletion and snf22+gcn5+ double deletion abolished chromatin remodeling and significant reduction of meiotic Recombination around M26. These results suggest that HATs and ADCRs cooperatively alter local chromatin structure, as in selective transcription activation, to activate meiotic Recombination at M26 in a site-specific manner.
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fission yeast tup1 like repressors repress chromatin remodeling at the fbp1 promoter and the ade6 m26 Recombination Hotspot
Genetics, 2003Co-Authors: Kouji Hirota, Takehiko Shibata, Charles S Hoffman, Kunihiro OhtaAbstract:Chromatin remodeling plays crucial roles in the regulation of gene expression and Recombination. Transcription of the fission yeast fbp1(+) gene and Recombination at the meiotic Recombination Hotspot ade6-M26 (M26) are both regulated by cAMP responsive element (CRE)-like sequences and the CREB/ATF-type transcription factor Atf1*Pcr1. The Tup11 and Tup12 proteins, the fission yeast counterparts of the Saccharomyces cerevisiae Tup1 corepressor, are involved in glucose repression of the fbp1(+) transcription. We have analyzed roles of the Tup1-like corepressors in chromatin regulation around the fbp1(+) promoter and the M26 Hotspot. We found that the chromatin structure around two regulatory elements for fbp1(+) was remodeled under derepressed conditions in concert with the robust activation of fbp1(+) transcription. Strains with tup11delta tup12delta double deletions grown in repressed conditions exhibited the chromatin state associated with wild-type cells grown in derepressed conditions. Interestingly, deletion of rst2(+), encoding a transcription factor controlled by the cAMP-dependent kinase, alleviated the tup11delta tup12delta defects in chromatin regulation but not in transcription repression. The chromatin at the M26 site in mitotic cultures of a tup11delta tup12delta mutant resembled that of wild-type meiotic cells. These observations suggest that these fission yeast Tup1-like corepressors repress chromatin remodeling at CRE-related sequences and that Rst2 antagonizes this function.
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A 160-bp Palindrome Is a Rad50·Rad32-Dependent Mitotic Recombination Hotspot in Schizosaccharomyces pombe
Genetics, 2002Co-Authors: Joseph A. Farah, Kenichi Mizuno, Kunihiro Ohta, Edgar Hartsuiker, Gerald R SmithAbstract:Abstract Palindromic sequences can form hairpin and cruciform structures that pose a threat to genome integrity. We found that a 160-bp palindrome (an inverted repeat of 80 bp) conferred a mitotic Recombination Hotspot relative to a control nonpalindromic sequence when inserted into the ade6 gene of Schizosaccharomyces pombe. The Hotspot activity of the palindrome, but not the basal level of Recombination, was abolished by a rad50 deletion, by a rad50S “separation of function” mutation, or by a rad32-D25A mutation in the nuclease domain of the Rad32 protein, an Mre11 homolog. We propose that upon extrusion of the palindrome the Rad50·Rad32 nuclease complex recognizes and cleaves the secondary structure thus formed and generates a recombinogenic break in the DNA.
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A 160-bp palindrome is a Rad50.Rad32-dependent mitotic Recombination Hotspot in Schizosaccharomyces pombe
Genetics, 2002Co-Authors: Joseph A. Farah, Kenichi Mizuno, Kunihiro Ohta, Edgar Hartsuiker, Gerald R SmithAbstract:Palindromic sequences can form hairpin and cruciform structures that pose a threat to genome integrity. We found that a 160-bp palindrome (an inverted repeat of 80 bp) conferred a mitotic Recombination Hotspot relative to a control nonpalindromic sequence when inserted into the ade6 gene of Schizosaccharomyces pombe. The Hotspot activity of the palindrome, but not the basal level of Recombination, was abolished by a rad50 deletion, by a rad50S "separation of function" mutation, or by a rad32-D25A mutation in the nuclease domain of the Rad32 protein, an Mre11 homolog. We propose that upon extrusion of the palindrome the Rad50.Rad32 nuclease complex recognizes and cleaves the secondary structure thus formed and generates a recombinogenic break in the DNA.