scholarly journals Competition between the RNA Transcript and the Nontemplate DNA Strand during R-Loop Formation In Vitro: a Nick Can Serve as a Strong R-Loop Initiation Site

2009 ◽  
Vol 30 (1) ◽  
pp. 146-159 ◽  
Author(s):  
Deepankar Roy ◽  
Zheng Zhang ◽  
Zhengfei Lu ◽  
Chih-Lin Hsieh ◽  
Michael R. Lieber
Keyword(s):  
Somatic Hypermutation ◽  
Initiation Site ◽  
Loop Formation ◽  
Class Switch ◽  
Dna Strands ◽  
Dna Strand ◽  
Template Dna ◽  
Rna Transcript

ABSTRACT Upon transcription of some sequences by RNA polymerases in vitro or in vivo, the RNA transcript can thread back onto the template DNA strand, resulting in an R loop. Previously, we showed that initiation of R-loop formation at an R-loop initiation zone (RIZ) is favored by G clusters. Here, using a purified in vitro system with T7 RNA polymerase, we show that increased distance between the promoter and the R-loop-supporting G-rich region reduces R-loop formation. When the G-rich portion of the RNA transcript is downstream from the 5′ end of the transcript, the ability of this portion of the transcript to anneal to the template DNA strand is reduced. When we nucleolytically resect the beginning of the transcript, R-loop formation increases because the G-rich portion of the RNA is now closer to the 5′ end of the transcript. Short G-clustered regions can act as RIZs and reduce the distance-induced suppression of R-loop formation. Supercoiled DNA is known to favor transient separation of the two DNA strands, and we find that this favors R-loop formation even in non-G-rich regions. Most strikingly, a nick can serve as a strong RIZ, even in regions with no G richness. This has important implications for class switch recombination and somatic hypermutation and possibly for other biological processes in transcribed regions.

scholarly journals The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription

2009 ◽  
Vol 206 (5) ◽  
pp. 1057-1071 ◽  
Author(s):  
Hong Ming Shen ◽  
Michael G. Poirier ◽  
Michael J. Allen ◽  
Justin North ◽  
Ratnesh Lal ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Naked Dna ◽  
Class Switch ◽  
Activation Induced Cytidine Deaminase ◽  
Dna Strands ◽  
Ampicillin Resistance Gene ◽  
Cytidine Deamination

The activation-induced cytidine deaminase (AID) initiates somatic hypermutation, class-switch recombination, and gene conversion of immunoglobulin genes. In vitro, AID has been shown to target single-stranded DNA, relaxed double-stranded DNA, when transcribed, or supercoiled DNA. To simulate the in vivo situation more closely, we have introduced two copies of a nucleosome positioning sequence, MP2, into a supercoiled AID target plasmid to determine where around the positioned nucleosomes (in the vicinity of an ampicillin resistance gene) cytidine deaminations occur in the absence or presence of transcription. We found that without transcription nucleosomes prevented cytidine deamination by AID. However, with transcription AID readily accessed DNA in nucleosomes on both DNA strands. The experiments also showed that AID targeting any DNA molecule was the limiting step, and they support the conclusion that once targeted to DNA, AID acts processively in naked DNA and DNA organized within transcribed nucleosomes.

scholarly journals Targeting of the Activation-Induced Cytosine Deaminase Is Strongly Influenced by the Sequence and Structure of the Targeted DNA

2005 ◽  
Vol 25 (24) ◽  
pp. 10815-10821 ◽  
Author(s):  
Hong Ming Shen ◽  
Sarayu Ratnam ◽  
Ursula Storb
Keyword(s):  
Dna Sequence ◽  
Somatic Hypermutation ◽  
Cytosine Deaminase ◽  
Target Sequence ◽  
Cytosine Deamination ◽  
Dna Strands ◽  
Activation Induced Deaminase ◽  
Dna Strand

ABSTRACT Activation-induced deaminase (AID) initiates immunoglobulin somatic hypermutation (SHM). Since in vitro AID was shown to deaminate cytosines on single-stranded DNA or the nontranscribed strand, it remained a puzzle how in vivo AID targets both DNA strands equally. Here we investigate the roles of transcription and DNA sequence in cytosine deamination. Strikingly different results are found with different substrates. Depending on the target sequence, the transcribed DNA strand is targeted as well as or better than the nontranscribed strand. The preferential targeting is not related to the frequency of AID hot spots. Comparison of cytosine deamination by AID and bisulfite shows different targeting patterns suggesting that AID may locally unwind the DNA. We conclude that somatic hypermutation on both DNA strands is the natural outcome of AID action on a transcribed gene; furthermore, the DNA sequence or structure and topology play major roles in targeting AID in vitro and in vivo. On the other hand, the lack of mutations in the first ∼100 nucleotides and beyond about 1 to 2 kb from the promoter of immunoglobulin genes during SHM must be due to special conditions of transcription and chromatin in vivo.

scholarly journals The FLP recombinase of the Saccharomyces cerevisiae 2 microns plasmid attaches covalently to DNA via a phosphotyrosyl linkage.

1985 ◽  
Vol 5 (11) ◽  
pp. 3274-3279 ◽  
Author(s):  
R M Gronostajski ◽  
P D Sadowski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Flp Recombinase ◽  
Specific Target ◽  
Target Sites ◽  
Dna Strands ◽  
2 Micron Plasmid ◽  
Dna Strand

The FLP recombinase, encoded by the 2 micron plasmid of Saccharomyces cerevisiae, promotes efficient recombination in vivo and in vitro between its specific target sites (FLP sites). It was previously determined that FLP interacts with DNA sequences within its target site (B. J. Andrews, G. A. Proteau, L. G. Beatty, and P. D. Sadowski. Cell 40:795-803, 1985), generates a single-stranded break on both DNA strands within the FLP site, and remains covalently attached to the 3' end of each break. We now show that the FLP protein is bound to the 3' side of each break by an O-phosphotyrosyl residue and that it appears that the same tyrosyl residue(s) is used to attach to either DNA strand within the FLP site.

scholarly journals A Role for Msh6 But Not Msh3 in Somatic Hypermutation and Class Switch Recombination

2004 ◽  
Vol 200 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Stella A. Martomo ◽  
William W. Yang ◽  
Patricia J. Gearhart
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Wild Type ◽  
Class Switch ◽  
Entry Points ◽  
Limited Entry ◽  
Dna Strands ◽  
V Genes ◽  
Deficient Mice

Somatic hypermutation is initiated by activation-induced cytidine deaminase (AID), and occurs in several kilobases of DNA around rearranged immunoglobulin variable (V) genes and switch (S) sites before constant genes. AID deaminates cytosine to uracil, which can produce mutations of C:G nucleotide pairs, and the mismatch repair protein Msh2 participates in generating substitutions of downstream A:T pairs. Msh2 is always found as a heterodimer with either Msh3 or Msh6, so it is important to know which one is involved. Therefore, we sequenced V and S regions from Msh3- and Msh6-deficient mice and compared mutations to those from wild-type mice. Msh6-deficient mice had fewer substitutions of A and T bases in both regions and reduced heavy chain class switching, whereas Msh3-deficient mice had normal antibody responses. This establishes a role for the Msh2-Msh6 heterodimer in hypermutation and switch recombination. When the positions of mutation were mapped, several focused peaks were found in Msh6−/− clones, whereas mutations were dispersed in Msh3−/− and wild-type clones. The peaks occurred at either G or C in WGCW motifs (W = A or T), indicating that C was mutated on both DNA strands. This suggests that AID has limited entry points into V and S regions in vivo, and subsequent mutation requires Msh2-Msh6 and DNA polymerase.

scholarly journals The affinity of the S9.6 antibody for double-stranded RNAs impacts the mapping of R-loops in fission yeast

2017 ◽  
Author(s):  
Stella R. Hartono ◽  
Amélie Malapert ◽  
Pénélope Legros ◽  
Pascal Bernard ◽  
Frédéric Chédin ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Rnase H ◽  
Loop Formation ◽  
Rnase Iii ◽  
Genome Wide ◽  
Dna Strands ◽  
Nucleotide Resolution ◽  
Experimental Strategies

ABSTRACTR-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions.

The FLP recombinase of the Saccharomyces cerevisiae 2 microns plasmid attaches covalently to DNA via a phosphotyrosyl linkage

1985 ◽  
Vol 5 (11) ◽  
pp. 3274-3279
Author(s):  
R M Gronostajski ◽  
P D Sadowski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Flp Recombinase ◽  
Specific Target ◽  
Target Sites ◽  
Dna Strands ◽  
2 Micron Plasmid ◽  
Dna Strand

The FLP recombinase, encoded by the 2 micron plasmid of Saccharomyces cerevisiae, promotes efficient recombination in vivo and in vitro between its specific target sites (FLP sites). It was previously determined that FLP interacts with DNA sequences within its target site (B. J. Andrews, G. A. Proteau, L. G. Beatty, and P. D. Sadowski. Cell 40:795-803, 1985), generates a single-stranded break on both DNA strands within the FLP site, and remains covalently attached to the 3' end of each break. We now show that the FLP protein is bound to the 3' side of each break by an O-phosphotyrosyl residue and that it appears that the same tyrosyl residue(s) is used to attach to either DNA strand within the FLP site.

scholarly journals In vivo and in vitro studies of immunoglobulin gene somatic hypermutation

2001 ◽  
Vol 356 (1405) ◽  
pp. 21-28 ◽  
Author(s):  
Julian E. Sale ◽  
Mats Bemark ◽  
Gareth T. Williams ◽  
Christopher J. Jolly ◽  
Michael R. Ehrenstein ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Class Switch Recombination ◽  
Variable Region ◽  
Immunoglobulin Gene ◽  
Immunoglobulin Genes ◽  
Class Switch ◽  
Immunoglobulin Repertoire ◽  
Genetic Events

Following antigen encounter, two distinct processes modify immunoglobulin genes. The variable region is diversified by somatic hypermutation while the constant region may be changed by class–switch recombination. Although both genetic events can occur concurrently within germinal centre B cells, there are examples of each occurring independently of the other. Here we compare the contributions of class–switch recombination and somatic hypermutation to the diversification of the serum immunoglobulin repertoire and review evidence that suggests that, despite clear differences, the two processes may share some aspects of their mechanism in common.

scholarly journals G Clustering Is Important for the Initiation of Transcription-Induced R-Loops In Vitro, whereas High G Density without Clustering Is Sufficient Thereafter

2009 ◽  
Vol 29 (11) ◽  
pp. 3124-3133 ◽  
Author(s):  
Deepankar Roy ◽  
Michael R. Lieber
Keyword(s):  
Rna Polymerase ◽  
Cytidine Deaminase ◽  
Loop Formation ◽  
Initiation Phase ◽  
Initiation Zone ◽  
Loop Region ◽  
Switch Regions ◽  
Dna Strand

ABSTRACT R-loops form cotranscriptionally in vitro and in vivo at transcribed duplex DNA regions when the nascent RNA is G-rich, particularly with G clusters. This is the case for phage polymerases, as used here (T7 RNA polymerase), as well as RNA polymerases in bacteria, Saccharomyces cerevisiae, avians, mice, and humans. The nontemplate strand is left in a single-stranded configuration within the R-loop region. These structures are known to form at mammalian immunoglobulin class switch regions, thus exposing regions of single-stranded DNA for the action of AID, a single-strand-specific cytidine deaminase. R-loops form by thread-back of the RNA onto the template DNA strand, and here we report that G clusters are extremely important for the initiation phase of R-loop formation. Even very short regions with one GGGG sequence can initiate R-loops much more efficiently than random sequences. The high efficiencies observed with G clusters cannot be achieved by having a very high G density alone. Annealing of the transcript, which is otherwise disadvantaged relative to the nontemplate DNA strand because of unfavorable proximity while exiting the RNA polymerase, can offer greater stability if it occurs at the G clusters, thereby initiating an R-loop. R-loop elongation beyond the initiation zone occurs in a manner that is not as reliant on G clusters as it is on a high G density. These results lead to a model in which G clusters are important to nucleate the thread-back of RNA for R-loop initiation and, once initiated, the elongation of R-loops is primarily determined by the density of G on the nontemplate DNA strand. Without both a favorable R-loop initiation zone and elongation zone, R-loop formation is inefficient.

scholarly journals Human PMS2 deficiency is associated with impaired immunoglobulin class switch recombination

2008 ◽  
Vol 205 (11) ◽  
pp. 2465-2472 ◽  
Author(s):  
Sophie Péron ◽  
Ayse Metin ◽  
Pauline Gardès ◽  
Marie-Alexandra Alyanakian ◽  
Eamonn Sheridan ◽  
...  
Keyword(s):  
Somatic Hypermutation ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Class Switch ◽  
Double Strand Dna Breaks ◽  
Switch Regions

Immunoglobulin (Ig) class switch recombination (CSR) deficiencies are rare primary immunodeficiencies characterized by the lack of switched isotype (IgG/IgA/IgE) production. In some cases, CSR deficiencies can be associated with abnormal somatic hypermutation. Analysis of CSR deficiencies has helped reveal the key functions of CSR-triggering molecules, i.e., CD40L, CD40, and effector molecules such as activation-induced cytidine deaminase and uracil N-glycosylase. We report a new form of B cell–intrinsic CSR deficiency found in three patients with deleterious, homozygous mutations in the gene encoding the PMS2 component of the mismatch repair machinery. CSR was found partially defective in vivo and markedly impaired in vitro. It is characterized by the defective occurrence of double-strand DNA breaks (DSBs) in switch regions and abnormal formation of switch junctions. This observation strongly suggests a role for PMS2 in CSR-induced DSB generation.

scholarly journals Mechanism of R-Loop Formation at Immunoglobulin Class Switch Sequences

2007 ◽  
Vol 28 (1) ◽  
pp. 50-60 ◽  
Author(s):  
Deepankar Roy ◽  
Kefei Yu ◽  
Michael R. Lieber
Keyword(s):  
Critical Level ◽  
Biochemical Mechanism ◽  
Loop Formation ◽  
Independent Basis ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Switch Regions ◽  
Dna Strand ◽  
Formation Efficiency

ABSTRACT R-loops have been described in vivo at the immunoglobulin class switch sequences and at prokaryotic and mitochondrial origins of replication. However, the biochemical mechanism and determinants of R-loop formation are unclear. We find that R-loop formation is nearly eliminated when RNase T1 is added during transcription but not when it is added afterward. Hence, rather than forming simply as an extension of the RNA-DNA hybrid of normal transcription, the RNA must exit the RNA polymerase and compete with the nontemplate DNA strand for an R-loop to form. R-loops persist even when transcription is done in Li+ or Cs+, which do not support G-quartet formation. Hence, R-loop formation does not rely on G-quartet formation. R-loop formation efficiency decreases as the number of switch repeats is decreased, although a very low level of R-loop formation occurs at even one 49-bp switch repeat. R-loop formation decreases sharply as G clustering is reduced, even when G density is kept constant. The critical level for R-loop formation is approximately the same point to which evolution drove the G clustering and G density on the nontemplate strand of mammalian switch regions. This provides an independent basis for concluding that the primary function of G clustering, in the context of high G density, is R-loop formation.

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