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.

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 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.

Mechanism of R‐Loop formation at Immunoglobulin Class Switch sequences

2008 ◽  
Vol 22 (S2) ◽  
pp. 416-416
Author(s):  
Deepankar Roy ◽  
Kefei Yu ◽  
Michael R Lieber
Keyword(s):  
Loop Formation ◽  
Immunoglobulin Class ◽  
Class Switch

scholarly journals Fine-Structure Analysis of Activation-Induced Deaminase Accessibility to Class Switch Region R-Loops

2005 ◽  
Vol 25 (5) ◽  
pp. 1730-1736 ◽  
Author(s):  
Kefei Yu ◽  
Deepankar Roy ◽  
Melina Bayramyan ◽  
Ian S. Haworth ◽  
Michael R. Lieber
Keyword(s):  
Somatic Hypermutation ◽  
Class Switch Recombination ◽  
Site Preference ◽  
Switch Region ◽  
Class Switch ◽  
High Resolution Analysis ◽  
Switch Regions ◽  
Resolution Analysis ◽  
Activation Induced Deaminase ◽  
Dna Strand

ABSTRACT Activation-induced deaminase (AID) is essential for class switch recombination and somatic hypermutation, and it has the ability to deaminate single-stranded DNA at cytidines. Mammalian class switch regions form R-loops upon transcription in the physiological orientation. The displaced DNA strand of an R-loop is forced to wrap around the RNA-DNA hybrid; hence, it may not have complete exposure to proteins. A fundamental question concerns the extent to which AID is accessible to the displaced strand of a transcription-generated R-loop. We used a minimal R-loop to carry out high-resolution analysis of the precise locations of AID action. We found that AID deaminates on the displaced DNA strand across the entire length of the R-loop. Displaced strand locations with a WRC (where W is A or T and R is A or G) sequence are preferred targets, but there are clear exceptions. These WRC deviations may be due to steric constraints on the accessibility of AID to these sites as the displaced strand twists around the RNA-DNA duplex. This phenomenon may explain the lack of WRC site preference at the mutations surrounding class switch recombination junctions.

R-loops at immunoglobulin class switch regions in the chromosomes of stimulated B cells

10.1038/ni919 ◽  
2003 ◽  
Vol 4 (5) ◽  
pp. 442-451 ◽  
Author(s):  
Kefei Yu ◽  
Frederic Chedin ◽  
Chih-Lin Hsieh ◽  
Thomas E. Wilson ◽  
Michael R. Lieber
Keyword(s):  
B Cells ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Switch Regions

scholarly journals Parp1 facilitates alternative NHEJ, whereas Parp2 suppresses IgH/c-myc translocations during immunoglobulin class switch recombination

2009 ◽  
Vol 206 (5) ◽  
pp. 1047-1056 ◽  
Author(s):  
Isabelle Robert ◽  
Françoise Dantzer ◽  
Bernardo Reina-San-Martin
Keyword(s):  
Dna Damage ◽  
Cytidine Deaminase ◽  
Class Switch Recombination ◽  
Dependent Manner ◽  
Dna Breaks ◽  
End Joining ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Switch Regions ◽  
Alternative Nhej

Immunoglobulin class switch recombination (CSR) is initiated by DNA breaks triggered by activation-induced cytidine deaminase (AID). These breaks activate DNA damage response proteins to promote appropriate repair and long-range recombination. Aberrant processing of these breaks, however, results in decreased CSR and/or increased frequency of illegitimate recombination between the immunoglobulin heavy chain locus and oncogenes like c-myc. Here, we have examined the contribution of the DNA damage sensors Parp1 and Parp2 in the resolution of AID-induced DNA breaks during CSR. We find that although Parp enzymatic activity is induced in an AID-dependent manner during CSR, neither Parp1 nor Parp2 are required for CSR. We find however, that Parp1 favors repair of switch regions through a microhomology-mediated pathway and that Parp2 actively suppresses IgH/c-myc translocations. Thus, we define Parp1 as facilitating alternative end-joining and Parp2 as a novel translocation suppressor during CSR.

scholarly journals Ongoing In Vivo Immunoglobulin Class Switch DNA Recombination in Chronic Lymphocytic Leukemia B Cells

2002 ◽  
Vol 169 (11) ◽  
pp. 6594-6603 ◽  
Author(s):  
Andrea Cerutti ◽  
Hong Zan ◽  
Edmund C. Kim ◽  
Shefali Shah ◽  
Elaine J. Schattner ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Dna Recombination ◽  
Lymphocytic Leukemia ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Class Switch Dna Recombination

scholarly journals Rev1 Recruits Ung to Switch Regions and Enhances dU Glycosylation for Immunoglobulin Class Switch DNA Recombination

Cell Reports ◽  
2012 ◽  
Vol 2 (5) ◽  
pp. 1220-1232 ◽  
Author(s):  
Hong Zan ◽  
Clayton A. White ◽  
Lisa M. Thomas ◽  
Thach Mai ◽  
Guideng Li ◽  
...  
Keyword(s):  
Dna Recombination ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Switch Regions ◽  
Class Switch Dna Recombination

scholarly journals Role for DNA repair factor XRCC4 in immunoglobulin class switch recombination

2007 ◽  
Vol 204 (7) ◽  
pp. 1717-1727 ◽  
Author(s):  
Pauline Soulas-Sprauel ◽  
Gwenaël Le Guyader ◽  
Paola Rivera-Munoz ◽  
Vincent Abramowski ◽  
Christelle Olivier-Martin ◽  
...  
Keyword(s):  
Dna Repair ◽  
B Lymphocyte ◽  
Alternative Pathway ◽  
Class Switch Recombination ◽  
Immunoglobulin Class ◽  
Class Switch ◽  
Lentiviral Transgenesis ◽  
Repair Factor

V(D)J recombination and immunoglobulin class switch recombination (CSR) are two somatic rearrangement mechanisms that proceed through the introduction of double-strand breaks (DSBs) in DNA. Although the DNA repair factor XRCC4 is essential for the resolution of DNA DSB during V(D)J recombination, its role in CSR has not been established. To bypass the embryonic lethality of XRCC4 deletion in mice, we developed a conditional XRCC4 knockout (KO) using LoxP-flanked XRCC4 cDNA lentiviral transgenesis. B lymphocyte restricted deletion of XRCC4 in these mice lead to an average two-fold reduction in CSR in vivo and in vitro. Our results connect XRCC4 and the nonhomologous end joining DNA repair pathway to CSR while reflecting the possible use of an alternative pathway in the repair of CSR DSB in the absence of XRCC4. In addition, this new conditional KO approach should be useful in studying other lethal mutations in mice.

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