scholarly journals Synapsis of Recombination Signal Sequences Located in cis and DNA Underwinding in V(D)J Recombination

2004 ◽  
Vol 24 (19) ◽  
pp. 8727-8744 ◽  
Author(s):  
Mihai Ciubotaru ◽  
David G. Schatz
Keyword(s):  
Conformational Changes ◽  
Dna Cleavage ◽  
High Mobility ◽  
Experimental System ◽  
High Mobility Group Protein ◽  
Signal Sequences ◽  
Recombination Signal ◽  
Recombination Signal Sequences ◽  
Group Protein ◽  
Dna Substrates

ABSTRACT V(D)J recombination requires binding and synapsis of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins, aided by a high-mobility group protein, HMG1 or HMG2. Double-strand DNA cleavage within this synaptic, or paired, complex is thought to involve DNA distortion or melting near the site of cleavage. Although V(D)J recombination normally occurs between RSSs located on the same DNA molecule (in cis), all previous studies that directly assessed RSS synapsis were performed with the two DNA substrates in trans. To overcome this limitation, we have developed a facilitated circularization assay using DNA substrates of reduced length to assess synapsis of RSSs in cis. We show that a 12/23 pair of RSSs is the preferred substrate for synapsis of cis RSSs and that the efficiency of pairing is dependent upon RAG1-RAG2 stoichiometry. Synapsis in cis occurs rapidly and is kinetically favored over synapsis of RSSs located in trans. This experimental system also allowed the generation of underwound DNA substrates containing pairs of RSSs in cis. Importantly, we found that the RAG proteins cleave such substrates substantially more efficiently than relaxed substrates and that underwinding may enhance RSS synapsis as well as RAG1/2-mediated catalysis. The energy stored in such underwound substrates may be used in the generation of DNA distortion and/or protein conformational changes needed for synapsis and cleavage. We propose that this unwinding is uniquely sensed during synapsis of an appropriate 12/23 pair of RSSs.

scholarly journals Fluorescence Resonance Energy Transfer Analysis of Recombination Signal Sequence Configuration in the RAG1/2 Synaptic Complex

2007 ◽  
Vol 27 (13) ◽  
pp. 4745-4758 ◽  
Author(s):  
Mihai Ciubotaru ◽  
Aleksei N. Kriatchko ◽  
Patrick C. Swanson ◽  
Frank V. Bright ◽  
David G. Schatz
Keyword(s):  
Energy Transfer ◽  
Metal Ion ◽  
Signal Sequence ◽  
Resonance Energy Transfer ◽  
High Mobility ◽  
Resonance Energy ◽  
High Mobility Group Protein ◽  
Synaptic Complex ◽  
Recombination Signal ◽  
Group Protein

ABSTRACT A critical step in V(D)J recombination is the synapsis of complementary (12/23) recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins to generate the paired complex (PC). Using a facilitated ligation assay and substrates that vary the helical phasing of the RSSs, we provide evidence that one particular geometric configuration of the RSSs is favored in the PC. To investigate this configuration further, we used fluorescent resonance energy transfer (FRET) to detect the synapsis of fluorescently labeled RSS oligonucleotides. FRET requires an appropriate 12/23 RSS pair, a divalent metal ion, and high-mobility-group protein HMGB1 or HMGB2. Energy transfer between the RSSs was detected with all 12/23 RSS end positions of the fluorescent probes but was not detected when probes were placed on the two ends of the same RSS. Energy transfer was confirmed to originate from the PC by using an in-gel FRET assay. The results argue against a unique planar configuration of the RSSs in the PC and are most easily accommodated by models in which synapsed 12- and 23-RSSs are bent and cross one another, with implications for the organization of the RAG proteins and the DNA substrates at the time of cleavage.

scholarly journals Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination

2015 ◽  
Vol 35 (21) ◽  
pp. 3701-3713 ◽  
Author(s):  
Keerthi Shetty ◽  
David G. Schatz
Keyword(s):  
Cell Lines ◽  
Chromatin Immunoprecipitation ◽  
Dna Cleavage ◽  
Time Course ◽  
Mutant Form ◽  
Signal Sequences ◽  
Recombination Signal ◽  
Recombination Signal Sequences ◽  
Cryptic Rss ◽  
Retroviral Recombination

V(D)J recombination is initiated by the binding of the RAG1 and RAG2 proteins to recombination signal sequences (RSSs) that consist of conserved heptamer and nonamer sequences separated by a spacer of either 12 or 23 bp. Here, we used RAG-inducible pro-B v-Abl cell lines in conjunction with chromatin immunoprecipitation to better understand the protein and RSS requirements for RAG recruitment to chromatin. Using a catalytic mutant form of RAG1 to prevent recombination, we did not observe cooperation between RAG1 and RAG2 in their recruitment to endogenous Jκ gene segments over a 48-h time course. Using retroviral recombination substrates, we found that RAG1 was recruited inefficiently to substrates lacking an RSS or containing a single RSS, better to substrates with two 12-bp RSSs (12RSSs) or two 23-bp RSSs (23RSSs), and more efficiently to a substrate with a 12/23RSS pair. RSS mutagenesis demonstrated a major role for the nonamer element in RAG1 binding, and correspondingly, a cryptic RSS consisting of a repeat of CA dinucleotides, which poorly re-creates the nonamer, was ineffective in recruiting RAG1. Our findings suggest that 12RSS-23RSS cooperation (the “12/23 rule”) is important not only for regulating RAG-mediated DNA cleavage but also for the efficiency of RAG recruitment to chromatin.

scholarly journals The RAG1 Homeodomain Recruits HMG1 and HMG2 To Facilitate Recombination Signal Sequence Binding and To Enhance the Intrinsic DNA-Bending Activity of RAG1-RAG2

1999 ◽  
Vol 19 (10) ◽  
pp. 6532-6542 ◽  
Author(s):  
Vassilis Aidinis ◽  
Tiziana Bonaldi ◽  
Monica Beltrame ◽  
Sandro Santagata ◽  
Marco E. Bianchi ◽  
...  
Keyword(s):  
Signal Sequence ◽  
Specific Binding ◽  
High Mobility ◽  
Recombination Reaction ◽  
High Mobility Group Protein ◽  
Bent Dna ◽  
Recombination Signal ◽  
Group Protein

ABSTRACT V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.

scholarly journals HMGA1 Is a Potential Driver of Preeclampsia Pathogenesis by Interference with Extravillous Trophoblasts Invasion

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 822
Author(s):  
Keiichi Matsubara ◽  
Yuko Matsubara ◽  
Yuka Uchikura ◽  
Katsuko Takagi ◽  
Akiko Yano ◽  
...  
Keyword(s):  
Protein A ◽  
High Mobility ◽  
Extravillous Trophoblast ◽  
High Mobility Group Protein ◽  
Successful Pregnancy ◽  
Group Protein ◽  
Serious Disease ◽  
Proliferation And Invasion ◽  
Extravillous Trophoblasts ◽  
Fertilized Egg

Preeclampsia (PE) is a serious disease that can be fatal for the mother and fetus. The two-stage theory has been proposed as its cause, with the first stage comprising poor placentation associated with the failure of fertilized egg implantation. Successful implantation and placentation require maternal immunotolerance of the fertilized egg as a semi-allograft and appropriate extravillous trophoblast (EVT) invasion of the decidua and myometrium. The disturbance of EVT invasion during implantation in PE results in impaired spiral artery remodeling. PE is thought to be caused by hypoxia during remodeling failure–derived poor placentation, which results in chronic inflammation. High-mobility group protein A (HMGA) is involved in the growth and invasion of cancer cells and likely in the growth and invasion of trophoblasts. Its mechanism of action is associated with immunotolerance. Thus, HMGA is thought to play a pivotal role in successful pregnancy, and its dysfunction may be related to the pathogenesis of PE. The evaluation of HMGA function and its changes in PE might confirm that it is a reliable biomarker of PE and provide prospects for PE treatment through the induction of EVT proliferation and invasion during the implantation.

scholarly journals Characterization of high mobility group protein levels during spermatogenesis in the rat.

1984 ◽  
Vol 259 (14) ◽  
pp. 8840-8846
Author(s):  
L R Bucci ◽  
W A Brock ◽  
I L Goldknopf ◽  
M L Meistrich
Keyword(s):  
High Mobility ◽  
High Mobility Group ◽  
High Mobility Group Protein ◽  
Protein Levels ◽  
Group Protein
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