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Gene Conversion-Like Events in the Diversification of Human Rearranged IGHV3-23*01 Gene Sequences.

Duvvuri B, Wu GE - Front Immunol (2012)

Bottom Line: We did not observe GCV-like events in rearranged IGHV3-23-(*)01 sequences from AID-deficient patients.GCV, unlike somatic hypermutation (SHM), can result in multiple base substitutions that can alter many amino acids.The extensive changes in antibody affinity by GCV-like events would be instrumental in protecting humans against pathogens that diversify their genome by antigenic shift.

View Article: PubMed Central - PubMed

Affiliation: School of Kinesiology and Health Science, Faculty of Health, York University Toronto, ON, Canada.

ABSTRACT
Gene conversion (GCV), a mechanism mediated by activation-induced cytidine deaminase (AID) is well established as a mechanism of immunoglobulin diversification in a few species. However, definitive evidence of GCV-like events in human immunoglobulin genes is scarce. The lack of evidence of GCV in human rearranged immunoglobulin gene sequences is puzzling given the presence of highly similar germline donors and the presence of all the enzymatic machinery required for GCV. In this study, we undertook a computational analysis of rearranged IGHV3-23(*)01 gene sequences from common variable immunodeficiency (CVID) patients, AID-deficient patients, and healthy individuals to survey "GCV-like" activities. We analyzed rearranged IGHV3-23(*)01 gene sequences obtained from total PBMC RNA and single-cell polymerase chain reaction of individual B cell lysates. Our search identified strong evidence of GCV-like activity. We observed that GCV-like tracts are flanked by AID hotspot motifs. Structural modeling of IGHV3-23(*)01 gene sequence revealed that hypermutable bases flanking GCV-like tracts are in the single stranded DNA (ssDNA) of stable stem-loop structures (SLSs). ssDNA is inherently fragile and also an optimal target for AID. We speculate that GCV could have been initiated by the targeting of hypermutable bases in ssDNA state in stable SLSs, plausibly by AID. We have observed that the frequency of GCV-like events is significantly higher in rearranged IGHV3-23-(*)01 sequences from healthy individuals compared to that of CVID patients. We did not observe GCV-like events in rearranged IGHV3-23-(*)01 sequences from AID-deficient patients. GCV, unlike somatic hypermutation (SHM), can result in multiple base substitutions that can alter many amino acids. The extensive changes in antibody affinity by GCV-like events would be instrumental in protecting humans against pathogens that diversify their genome by antigenic shift.

No MeSH data available.


Related in: MedlinePlus

Stem-loop structures of hypermutable bases in SHM/AID hotspot motifs flanking 5′ and 3′of GCV-like tracts. DNA secondary structures formed by folding germline IGHV3-23 sequence in program “mfg” during simulated transcription (Wright et al., 2003, 2004, 2008a,b). Each structure flanking GCV-like tracts are named as the rearranged IGHV3-23*01 sequences shown in Figures 1–4. The hypermutable bases are shown with arrows. DNA secondary structural parameters, −ΔG, % Unpaired (%UP) correspond to hypermutable base (shown with arrows).
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Figure 5: Stem-loop structures of hypermutable bases in SHM/AID hotspot motifs flanking 5′ and 3′of GCV-like tracts. DNA secondary structures formed by folding germline IGHV3-23 sequence in program “mfg” during simulated transcription (Wright et al., 2003, 2004, 2008a,b). Each structure flanking GCV-like tracts are named as the rearranged IGHV3-23*01 sequences shown in Figures 1–4. The hypermutable bases are shown with arrows. DNA secondary structural parameters, −ΔG, % Unpaired (%UP) correspond to hypermutable base (shown with arrows).

Mentions: With substantial evidence showing that RGYW/WRCY motifs are preferential targets of DSB’s and resected ends during SHM, we undertook structural analysis of bases that are present at or nearest to the break points of GCV-like tracts (5′ and 3′). The hypermutable bases in the flanking regions of GCV-like tracts for which structures are drawn are italicized in Figures 1–4. Since SHM (and AID) targeted bases are predominantly C’s and G’s, we analyzed the paired or unpaired state of C’s or G’s at or nearest to GCV-break points. We used the germline non-transcribed strand (NTS) of IGHV3-23*01 for the structural analysis through simulated transcription using the DNA secondary structural analysis tool, “mfg.” “Mfg” predicts the most stable SLS in which a particular base is unpaired. The propensity of a base to be in the single stranded condition is quantified as the percent unpaired (%UP) and the stabilities of the SLSs as −ΔGs’ are shown in Figure 5. Figure 5 represents the most stable SLSs in which hypermutable bases that are present at GCV-break points are exposed in unpaired or in ssDNA state (C’s or G’s, shown with arrows). This structural analysis allows the speculation that the observed GCV-like tracts in IGHV3-23*01 sequences are initiated by the DSB’s in the sequence-specific motifs owing to their localization in the ssDNA state in stable SLSs.


Gene Conversion-Like Events in the Diversification of Human Rearranged IGHV3-23*01 Gene Sequences.

Duvvuri B, Wu GE - Front Immunol (2012)

Stem-loop structures of hypermutable bases in SHM/AID hotspot motifs flanking 5′ and 3′of GCV-like tracts. DNA secondary structures formed by folding germline IGHV3-23 sequence in program “mfg” during simulated transcription (Wright et al., 2003, 2004, 2008a,b). Each structure flanking GCV-like tracts are named as the rearranged IGHV3-23*01 sequences shown in Figures 1–4. The hypermutable bases are shown with arrows. DNA secondary structural parameters, −ΔG, % Unpaired (%UP) correspond to hypermutable base (shown with arrows).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3375636&req=5

Figure 5: Stem-loop structures of hypermutable bases in SHM/AID hotspot motifs flanking 5′ and 3′of GCV-like tracts. DNA secondary structures formed by folding germline IGHV3-23 sequence in program “mfg” during simulated transcription (Wright et al., 2003, 2004, 2008a,b). Each structure flanking GCV-like tracts are named as the rearranged IGHV3-23*01 sequences shown in Figures 1–4. The hypermutable bases are shown with arrows. DNA secondary structural parameters, −ΔG, % Unpaired (%UP) correspond to hypermutable base (shown with arrows).
Mentions: With substantial evidence showing that RGYW/WRCY motifs are preferential targets of DSB’s and resected ends during SHM, we undertook structural analysis of bases that are present at or nearest to the break points of GCV-like tracts (5′ and 3′). The hypermutable bases in the flanking regions of GCV-like tracts for which structures are drawn are italicized in Figures 1–4. Since SHM (and AID) targeted bases are predominantly C’s and G’s, we analyzed the paired or unpaired state of C’s or G’s at or nearest to GCV-break points. We used the germline non-transcribed strand (NTS) of IGHV3-23*01 for the structural analysis through simulated transcription using the DNA secondary structural analysis tool, “mfg.” “Mfg” predicts the most stable SLS in which a particular base is unpaired. The propensity of a base to be in the single stranded condition is quantified as the percent unpaired (%UP) and the stabilities of the SLSs as −ΔGs’ are shown in Figure 5. Figure 5 represents the most stable SLSs in which hypermutable bases that are present at GCV-break points are exposed in unpaired or in ssDNA state (C’s or G’s, shown with arrows). This structural analysis allows the speculation that the observed GCV-like tracts in IGHV3-23*01 sequences are initiated by the DSB’s in the sequence-specific motifs owing to their localization in the ssDNA state in stable SLSs.

Bottom Line: We did not observe GCV-like events in rearranged IGHV3-23-(*)01 sequences from AID-deficient patients.GCV, unlike somatic hypermutation (SHM), can result in multiple base substitutions that can alter many amino acids.The extensive changes in antibody affinity by GCV-like events would be instrumental in protecting humans against pathogens that diversify their genome by antigenic shift.

View Article: PubMed Central - PubMed

Affiliation: School of Kinesiology and Health Science, Faculty of Health, York University Toronto, ON, Canada.

ABSTRACT
Gene conversion (GCV), a mechanism mediated by activation-induced cytidine deaminase (AID) is well established as a mechanism of immunoglobulin diversification in a few species. However, definitive evidence of GCV-like events in human immunoglobulin genes is scarce. The lack of evidence of GCV in human rearranged immunoglobulin gene sequences is puzzling given the presence of highly similar germline donors and the presence of all the enzymatic machinery required for GCV. In this study, we undertook a computational analysis of rearranged IGHV3-23(*)01 gene sequences from common variable immunodeficiency (CVID) patients, AID-deficient patients, and healthy individuals to survey "GCV-like" activities. We analyzed rearranged IGHV3-23(*)01 gene sequences obtained from total PBMC RNA and single-cell polymerase chain reaction of individual B cell lysates. Our search identified strong evidence of GCV-like activity. We observed that GCV-like tracts are flanked by AID hotspot motifs. Structural modeling of IGHV3-23(*)01 gene sequence revealed that hypermutable bases flanking GCV-like tracts are in the single stranded DNA (ssDNA) of stable stem-loop structures (SLSs). ssDNA is inherently fragile and also an optimal target for AID. We speculate that GCV could have been initiated by the targeting of hypermutable bases in ssDNA state in stable SLSs, plausibly by AID. We have observed that the frequency of GCV-like events is significantly higher in rearranged IGHV3-23-(*)01 sequences from healthy individuals compared to that of CVID patients. We did not observe GCV-like events in rearranged IGHV3-23-(*)01 sequences from AID-deficient patients. GCV, unlike somatic hypermutation (SHM), can result in multiple base substitutions that can alter many amino acids. The extensive changes in antibody affinity by GCV-like events would be instrumental in protecting humans against pathogens that diversify their genome by antigenic shift.

No MeSH data available.


Related in: MedlinePlus