Direct observation of RAG recombinase recruitment to chromatin and the IgH locus in live pro-B cells

AbstractThe RAG1 and RAG2 proteins introduce double-strand DNA breaks at antigen-receptor loci in developing lymphocytes to initiate V(D)J recombination. How RAG proteins find the correct target locus in a vast excess of non-specific chromatin is not known. Here we measured dynamics of RAG1/RAG2 interactions with chromatin in living pro-B cells. We found that the majority of RAG1 or RAG1/RAG2 complex is in a fast 3D diffusive state, and the residual slow diffusive (bound) fraction was determined by a non-core portion of RAG1, and the PHD domain of RAG2. The RAG proteins exhibited distinct dynamics at the IgH locus. In particular, RAG2 increased the probability of RAG1 binding to IgH, a property that likely explains its non-catalytic role in V(D)J recombination. Our observations reveal how RAG finds its targets in developing B cells.One Sentence SummarySingle-molecule imaging of the RAG recombinase reveals its search strategy for chromatin, H3K4me3 and antibody gene loci in living cells.

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TIN2 facilitates TRF1-mediated trans- and cis-interactions on physiologically relevant long telomeric DNA

10.1101/2020.09.12.286559 ◽

2020 ◽

Author(s):

Hai Pan ◽

Parminder Kaur ◽

Ming Liu ◽

Pengning Xu ◽

Chelsea Mahn ◽

...

Keyword(s):

Single Molecule ◽

Protein Complexes ◽

Molecular Structures ◽

Telomeric Sequence ◽

Dependent Manner ◽

Dna Breaks ◽

Telomeric Dna ◽

Double Strand Dna Breaks ◽

Regulator Protein ◽

Tankyrase 1

ABSTRACTThe shelterin complex consisting of TRF1, TRF2, RAP1, TIN2, TPP1, and POT1, functions to prevent false recognition of telomeres as double-strand DNA breaks, and to regulate telomerase and DNA repair protein access. TIN2 is a core component linking double-stranded telomeric DNA binding proteins (TRF1 and TRF2) and proteins at the 3’ overhang (TPP1-POT1). Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, determining TIN2’s unique mechanistic function has been elusive. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 using complementary single-molecule imaging platforms, including atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that TIN2S and TIN2L isoforms facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (6 TTAGGG repeats), the majority of TRF1 mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG), TIN2 forms multi-protein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last for at least minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 significantly reduces TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protects the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Our study provides evidence that TIN2 functions to promote TRF1 mediated trans-interactions of telomeric DNA, leading to new mechanistic insight into sister telomere cohesion.

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Genomic patterns of transcription-replication interactions in mouse primary B cells

10.1101/2021.04.13.439211 ◽

2021 ◽

Author(s):

Commodore P St Germain ◽

Hongchang Zhao ◽

Vrishti Sinha ◽

Lionel A Sanz ◽

Frederic Chedin ◽

...

Keyword(s):

B Cells ◽

Genome Stability ◽

Protein A ◽

Replication Stress ◽

Tumor Formation ◽

Dna Breaks ◽

Double Strand Dna Breaks ◽

Age Related ◽

Novel Method ◽

Genomic Locations

Conflicts between transcription and replication machinery are a potent source of replication stress and genome stability; however, no technique currently exists to identify endogenous genomic locations prone to transcription-replication interactions. Here, we report a novel method to identify genomic loci prone to transcription-replication interactions termed transcription-replication immunoprecipitation on nascent DNA sequencing, TRIPn-Seq. TRIPn-Seq employs the sequential immunoprecipitation of RNA polymerase 2 phosphorylated at serine 5 (RNAP2s5) followed by enrichment of nascent DNA previously labeled with bromodeoxyuridine. Using TRIPn-Seq, we mapped 1,009 unique transcription-replication interactions (TRIs) in mouse primary B cells characterized by a bimodal pattern of RNAP2s5, bidirectional transcription, an enrichment of RNA:DNA hybrids, and a high probability of forming G-quadruplexes. While TRIs themselves map to early replicating regions, they exhibit enhanced Replication Protein A association and replication fork termination, marks of replication stress. TRIs colocalize with double-strand DNA breaks, are enriched for deletions, and accumulate mutations in tumors. We propose that replication stress at TRIs induces mutations potentially contributing to age-related disease, as well as tumor formation and development.

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Hepatitis C Virus E2-CD81 Interaction Induces Hypermutation of the Immunoglobulin Gene in B Cells

Journal of Virology ◽

10.1128/jvi.79.13.8079-8089.2005 ◽

2005 ◽

Vol 79 (13) ◽

pp. 8079-8089 ◽

Cited By ~ 99

Author(s):

Keigo Machida ◽

Kevin T.-H. Cheng ◽

Nicole Pavio ◽

Vicky M.-H. Sung ◽

Michael M. C. Lai

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

B Cells ◽

B Cell ◽

B Cell Lymphoma ◽

Immunoglobulin Gene ◽

Dna Breaks ◽

Necrosis Factor Alpha ◽

Double Strand Dna Breaks ◽

Double Strand Dna

ABSTRACT Hepatitis C virus (HCV) is one of the leading causes of chronic liver diseases and B-lymphocyte proliferative disorders, including mixed cryoglobulinemia and B-cell lymphoma. It has been suggested that HCV infects human cells through the interaction of its envelope glycoprotein E2 with a tetraspanin molecule CD81, the putative viral receptor. Here, we show that the engagement of B cells by purified E2 induced double-strand DNA breaks specifically in the variable region of immunoglobulin (VH ) gene locus, leading to hypermutation in the VH genes of B cells. Other gene loci were not affected. Preincubation with the anti-CD81 monoclonal antibody blocked this effect. E2-CD81 interaction on B cells triggered the enhanced expression of activation-induced cytidine deaminase (AID) and also stimulated the production of tumor necrosis factor alpha. Knockdown of AID by the specific small interfering RNA blocked the E2-induced double-strand DNA breaks and hypermutation of the VH gene. These findings suggest that HCV infection, through E2-CD81 interaction, may modulate host's innate or adaptive immune response by activation of AID and hypermutation of immunoglobulin gene in B cells, leading to HCV-associated B-cell lymphoproliferative diseases.

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DNA barcoding reveals that injected transgenes are predominantly processed by homologous recombination in mouse zygote

Nucleic Acids Research ◽

10.1093/nar/gkz1085 ◽

2019 ◽

Cited By ~ 1

Author(s):

Alexander Smirnov ◽

Veniamin Fishman ◽

Anastasia Yunusova ◽

Alexey Korablev ◽

Irina Serova ◽

...

Keyword(s):

Homologous Recombination ◽

Single Molecule ◽

Strand Break ◽

Dna Molecules ◽

Dna Breaks ◽

Exogenous Dna ◽

Transgenic Organisms ◽

Double Strand Dna Breaks ◽

Strand Annealing ◽

Transgenic Embryos

Abstract Mechanisms that ensure repair of double-strand DNA breaks (DSBs) are instrumental in the integration of foreign DNA into the genome of transgenic organisms. After pronuclear microinjection, exogenous DNA is usually found as a concatemer comprising multiple co-integrated transgene copies. Here, we investigated the contribution of various DSB repair pathways to the concatemer formation. We injected mouse zygotes with a pool of linear DNA molecules carrying unique barcodes at both ends and obtained 10 transgenic embryos with 1–300 transgene copies. Sequencing the barcodes allowed us to assign relative positions to the copies in concatemers and detect recombination events that occurred during integration. Cumulative analysis of approximately 1,000 integrated copies reveals that over 80% of them underwent recombination when their linear ends were processed by synthesis-dependent strand annealing (SDSA) or double-strand break repair (DSBR). We also observed evidence of double Holliday junction (dHJ) formation and crossing over during the concatemer formations. Sequencing indels at the junctions between copies shows that at least 10% of DNA molecules introduced into the zygotes are ligated by non-homologous end joining (NHEJ). Our barcoding approach, verified with Pacific Biosciences Single Molecule Real-Time (SMRT) long-range sequencing, documents high activity of homologous recombination after DNA microinjection.

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TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA

Nucleic Acids Research ◽

10.1093/nar/gkab1142 ◽

2021 ◽

Author(s):

Parminder Kaur ◽

Ryan Barnes ◽

Hai Pan ◽

Ariana C Detwiler ◽

Ming Liu ◽

...

Keyword(s):

Single Molecule ◽

Molecular Model ◽

Loop Formation ◽

Dna Breaks ◽

Telomeric Dna ◽

Dna Structures ◽

Shelterin Complex ◽

Double Strand Dna Breaks ◽

Architectural Protein

Abstract The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2–TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein–DNA structures, and monitoring of DNA–DNA and DNA–RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA–dsDNA, dsDNA–ssDNA and dsDNA–ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.

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Prime-editors (nickases), hRad51–Cas9 nickase fusions and dCas9 have the same problem as conventional CRISPR-Cas9 of plasmid/Cas9 integration after making a double stranded break

10.31219/osf.io/jf6pe ◽

2019 ◽

Author(s):

Sandeep Chakraborty

Keyword(s):

Cellular Response ◽

Sequencing Data ◽

Dna Breaks ◽

Precise Integration ◽

Guide Rna ◽

Double Strand Dna Breaks ◽

Human Therapy ◽

P53 Activation ◽

Programmable Nucleases

‘Prime-editing’ proposes to replace traditional programmable nucleases (CRISPR-Cas9) using a catalytically impaired Cas9 (dCas9) connected to a engineered reverse transcriptase, and a guide RNA encoding both the target site and the desired change. With just a ‘nick’ on one strand, it is hypothe- sized, the negative, uncontrollable effects arising from double-strand DNA breaks (DSBs) - translocations, complex proteins, integrations and p53 activation - will be eliminated. However, sequencing data pro- vided (Accid:PRJNA565979) reveal plasmid integration, indicating that DSBs occur. Also, looking at only 16 off-targets is inadequate to assert that Prime-editing is more precise. Integration of plasmid occurs in all three versions (PE1/2/3). Interestingly, dCas9 which is known to be toxic in E. coli and yeast, is shown to have residual endonuclease activity. This also affects studies that use dCas9, like base- editors and de/methylations systems. Previous work using hRad51–Cas9 nickases also show significant integration in on-targets, as well as off-target integration [1]. Thus, we show that cellular response to nicking involves DSBs, and subsequent plasmid/Cas9 integration. This is an unacceptable outcome for any in vivo application in human therapy.

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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 ◽

10.1093/genetics/143.3.1115 ◽

1996 ◽

Vol 143 (3) ◽

pp. 1115-1125 ◽

Cited By ~ 3

Author(s):

Fei Xu ◽

Thomas D Petes

Keyword(s):

Meiotic Recombination ◽

Hydroxyl Groups ◽

Sequence Specificity ◽

Structure Mapping ◽

Dna Breaks ◽

Exonuclease Iii ◽

Recombination Hotspot ◽

Telomeric Sequences ◽

Double Strand Dna Breaks ◽

Double Strand Dna

Abstract 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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Competition Between Adjacent Meiotic Recombination Hotspots in the Yeast Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/145.3.661 ◽

1997 ◽

Vol 145 (3) ◽

pp. 661-670 ◽

Cited By ~ 1

Author(s):

Qing-Qing Fan ◽

Fei Xu ◽

Michael A White ◽

Thomas D Petes

Keyword(s):

Saccharomyces Cerevisiae ◽

Meiotic Recombination ◽

Telomeric Sequence ◽

Coding Region ◽

Dna Breaks ◽

Local Formation ◽

Yeast Saccharomyces Cerevisiae ◽

Recombination Hotspots ◽

Double Strand Dna Breaks ◽

His4 Gene

In a wild-type strain of Saccharomyces cerevisiae, a hotspot for meiotic recombination is located upstream of the HIS4 gene. An insertion of a 49-bp telomeric sequence into the coding region of HIS4 strongly stimulates meiotic recombination and the local formation of meiosis-specific double-strand DNA breaks (DSBs). When strains are constructed in which both hotspots are heterozygous, hotspot activity is substantially less when the hotspots are on the same chromosome than when they are on opposite chromosomes.

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