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Augmented expression of a human gene for 8-oxoguanine DNA glycosylase (MutM) in B lymphocytes of the dark zone in lymph node germinal centers.

Kuo FC, Sklar J - J. Exp. Med. (1997)

Bottom Line: Northern blot analysis indicated that the human gene is expressed as two alternatively spliced messenger RNAs within GC B cells at levels greatly exceeding that found in other tissues.In situ hybridization studies revealed that expression of this gene is most abundant within the dark zones of GCs.Both the function and localized expression of this gene suggest that it may play a role in somatic hypermutation of immunoglobulin genes.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
B cells that mediate normal, T cell-dependent, humoral immune responses must first pass through germinal centers (GCs) within the cortex of antigenically stimulated lymph nodes. As they move through the dark zone and then the light zone in the GC, B cells are subjected to somatic hypermutation and switch recombination within their rearranged immunoglobulin genes and also participate in a number of other processes that control development into memory cells or cells specialized for antibody secretion. To investigate the molecular mechanisms that contribute to B cell development within GCs, we constructed a recombinant DNA library enriched for cDNAs derived from human genes expressed in B cells at this site. This library was found to contain a cDNA structurally and functionally related to genes in bacteria and yeast for the DNA repair enzyme 8-oxoguanine DNA glycosylase. Northern blot analysis indicated that the human gene is expressed as two alternatively spliced messenger RNAs within GC B cells at levels greatly exceeding that found in other tissues. In situ hybridization studies revealed that expression of this gene is most abundant within the dark zones of GCs. Both the function and localized expression of this gene suggest that it may play a role in somatic hypermutation of immunoglobulin genes.

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Functional assays of the GST-GCN6 fusion protein. (A)  Trapping of the Schiff base intermediate by the addition of sodium borohydride. Double-stranded, 3′ end–labeled oligonucleotides either with  (oG:C, lanes 1–4) or without (G:C, lanes 5 and 6) an 8-oxoguanine at the  center of the fragment were incubated with either with GST alone (lanes  1 and 2) or GST-GCN6 fusion protein (lanes 3–6) in the presence (lanes  2, 4, and 6) or absence (lanes 1, 3, and 5) of sodium borohydride. The reaction products were analyzed by 8% SDS-PAGE gel electrophoresis followed by autoradiography. The presence of a protein–DNA conjugate  can be detected as a slow mobility band indicated by the arrow. (B)  Cleavage of double-stranded oligonucleotides containing 8-oxoguanine.  Double-stranded, 3′ end–labeled oligonucleotides with or without an  8-oxoguanine were incubated with GST alone (lanes 1 and 5) or GST-GCN6 fusion protein (lanes 3 and 6), and the reaction products analyzed  by electrophoresis in a 12% polyacrylamide-7M urea gel followed by autoradiography.
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Figure 5: Functional assays of the GST-GCN6 fusion protein. (A) Trapping of the Schiff base intermediate by the addition of sodium borohydride. Double-stranded, 3′ end–labeled oligonucleotides either with (oG:C, lanes 1–4) or without (G:C, lanes 5 and 6) an 8-oxoguanine at the center of the fragment were incubated with either with GST alone (lanes 1 and 2) or GST-GCN6 fusion protein (lanes 3–6) in the presence (lanes 2, 4, and 6) or absence (lanes 1, 3, and 5) of sodium borohydride. The reaction products were analyzed by 8% SDS-PAGE gel electrophoresis followed by autoradiography. The presence of a protein–DNA conjugate can be detected as a slow mobility band indicated by the arrow. (B) Cleavage of double-stranded oligonucleotides containing 8-oxoguanine. Double-stranded, 3′ end–labeled oligonucleotides with or without an 8-oxoguanine were incubated with GST alone (lanes 1 and 5) or GST-GCN6 fusion protein (lanes 3 and 6), and the reaction products analyzed by electrophoresis in a 12% polyacrylamide-7M urea gel followed by autoradiography.

Mentions: The yeast Ogg1 protein has been shown to possess an 8-oxoguanine DNA glycosylase activity (33). The glycosylase reaction was demonstrated to proceed through a Schiff base intermediate formed between the enzyme and the DNA at the site of the modified guanine. Furthermore, this intermediate could be trapped by addition of sodium borohydride (NaBH4) to the reaction (34). Given these properties of the yeast enzyme, we tested the ability of the protein encoded by the GCN6 cDNA to form in vitro the same intermediate as the yeast enzyme. A cDNA fusion construct containing all but the first 11 codons of the GCN6 coding sequence fused to sequence encoding GST was expressed in a strain of E. coli mutant for MutM (MC301), the gene for 8-oxoguanine DNA glycosylase in bacteria. Purified GST-GCN6 fusion protein was then incubated under a variety of conditions with end-labeled, double-stranded oligonucleotides with or without an oxidized guanine in one or the other strand at the center of the sequence. As shown in Fig. 5 A, a band representing a covalently linked protein– DNA complex was found only when oxidized guanine was included in the oligonucleotide substrate (Fig. 5 A, lanes 5 and 6) and only in the presence of NaBH4 (Fig. 5 A, lanes 3 and 4). The control GST protein alone produced no complex (Fig. 5 A, lanes 1 and 2). Additionally, the GST-GCN6 fusion protein caused nicking of the oligonucleotides at the position of the oxidized guanine in a manner similar to that observed with the yeast protein (Fig. 5 B).


Augmented expression of a human gene for 8-oxoguanine DNA glycosylase (MutM) in B lymphocytes of the dark zone in lymph node germinal centers.

Kuo FC, Sklar J - J. Exp. Med. (1997)

Functional assays of the GST-GCN6 fusion protein. (A)  Trapping of the Schiff base intermediate by the addition of sodium borohydride. Double-stranded, 3′ end–labeled oligonucleotides either with  (oG:C, lanes 1–4) or without (G:C, lanes 5 and 6) an 8-oxoguanine at the  center of the fragment were incubated with either with GST alone (lanes  1 and 2) or GST-GCN6 fusion protein (lanes 3–6) in the presence (lanes  2, 4, and 6) or absence (lanes 1, 3, and 5) of sodium borohydride. The reaction products were analyzed by 8% SDS-PAGE gel electrophoresis followed by autoradiography. The presence of a protein–DNA conjugate  can be detected as a slow mobility band indicated by the arrow. (B)  Cleavage of double-stranded oligonucleotides containing 8-oxoguanine.  Double-stranded, 3′ end–labeled oligonucleotides with or without an  8-oxoguanine were incubated with GST alone (lanes 1 and 5) or GST-GCN6 fusion protein (lanes 3 and 6), and the reaction products analyzed  by electrophoresis in a 12% polyacrylamide-7M urea gel followed by autoradiography.
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Related In: Results  -  Collection

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Figure 5: Functional assays of the GST-GCN6 fusion protein. (A) Trapping of the Schiff base intermediate by the addition of sodium borohydride. Double-stranded, 3′ end–labeled oligonucleotides either with (oG:C, lanes 1–4) or without (G:C, lanes 5 and 6) an 8-oxoguanine at the center of the fragment were incubated with either with GST alone (lanes 1 and 2) or GST-GCN6 fusion protein (lanes 3–6) in the presence (lanes 2, 4, and 6) or absence (lanes 1, 3, and 5) of sodium borohydride. The reaction products were analyzed by 8% SDS-PAGE gel electrophoresis followed by autoradiography. The presence of a protein–DNA conjugate can be detected as a slow mobility band indicated by the arrow. (B) Cleavage of double-stranded oligonucleotides containing 8-oxoguanine. Double-stranded, 3′ end–labeled oligonucleotides with or without an 8-oxoguanine were incubated with GST alone (lanes 1 and 5) or GST-GCN6 fusion protein (lanes 3 and 6), and the reaction products analyzed by electrophoresis in a 12% polyacrylamide-7M urea gel followed by autoradiography.
Mentions: The yeast Ogg1 protein has been shown to possess an 8-oxoguanine DNA glycosylase activity (33). The glycosylase reaction was demonstrated to proceed through a Schiff base intermediate formed between the enzyme and the DNA at the site of the modified guanine. Furthermore, this intermediate could be trapped by addition of sodium borohydride (NaBH4) to the reaction (34). Given these properties of the yeast enzyme, we tested the ability of the protein encoded by the GCN6 cDNA to form in vitro the same intermediate as the yeast enzyme. A cDNA fusion construct containing all but the first 11 codons of the GCN6 coding sequence fused to sequence encoding GST was expressed in a strain of E. coli mutant for MutM (MC301), the gene for 8-oxoguanine DNA glycosylase in bacteria. Purified GST-GCN6 fusion protein was then incubated under a variety of conditions with end-labeled, double-stranded oligonucleotides with or without an oxidized guanine in one or the other strand at the center of the sequence. As shown in Fig. 5 A, a band representing a covalently linked protein– DNA complex was found only when oxidized guanine was included in the oligonucleotide substrate (Fig. 5 A, lanes 5 and 6) and only in the presence of NaBH4 (Fig. 5 A, lanes 3 and 4). The control GST protein alone produced no complex (Fig. 5 A, lanes 1 and 2). Additionally, the GST-GCN6 fusion protein caused nicking of the oligonucleotides at the position of the oxidized guanine in a manner similar to that observed with the yeast protein (Fig. 5 B).

Bottom Line: Northern blot analysis indicated that the human gene is expressed as two alternatively spliced messenger RNAs within GC B cells at levels greatly exceeding that found in other tissues.In situ hybridization studies revealed that expression of this gene is most abundant within the dark zones of GCs.Both the function and localized expression of this gene suggest that it may play a role in somatic hypermutation of immunoglobulin genes.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Oncology, Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
B cells that mediate normal, T cell-dependent, humoral immune responses must first pass through germinal centers (GCs) within the cortex of antigenically stimulated lymph nodes. As they move through the dark zone and then the light zone in the GC, B cells are subjected to somatic hypermutation and switch recombination within their rearranged immunoglobulin genes and also participate in a number of other processes that control development into memory cells or cells specialized for antibody secretion. To investigate the molecular mechanisms that contribute to B cell development within GCs, we constructed a recombinant DNA library enriched for cDNAs derived from human genes expressed in B cells at this site. This library was found to contain a cDNA structurally and functionally related to genes in bacteria and yeast for the DNA repair enzyme 8-oxoguanine DNA glycosylase. Northern blot analysis indicated that the human gene is expressed as two alternatively spliced messenger RNAs within GC B cells at levels greatly exceeding that found in other tissues. In situ hybridization studies revealed that expression of this gene is most abundant within the dark zones of GCs. Both the function and localized expression of this gene suggest that it may play a role in somatic hypermutation of immunoglobulin genes.

Show MeSH
Related in: MedlinePlus