Nucleotide excision repair in an immunoglobulin variable gene is less efficient than in a housekeeping gene

Abstract

Immunoglobulin variable genes undergo several unusual genetic modifications to generate diversity, such as gene rearrangement, gene conversion, somatic hypermutation, and heavy chain class switch recombination. In view of these specialized processes, we examined the possibility that variable genes have intrinsic characteristics that allow them to be processed differently in the course of basic DNA transactions as well. This hypothesis was studied in an experimental system to gauge the relative efficiency of a DNA repair pathway, nucleotide excision repair, on a variable gene and a housekeeping gene. DNA damage was induced by ultraviolet light in murine hybridoma B cells, and repair was measured over time by an alkaline Southern blot technique, which detected removal of cyclobutane pyrimidine dimers. The rate of DNA repair in a rearranged variable gene, V(H)S107, was compared to that in the dihydrofolate reductase gene. Although both genes were actively transcribed, the V(H)S107 gene was repaired less efficiently than the dihydrofolate reductase gene. These results suggest that variable genes have inherent properties that affect the efficiency of nucleotide excision repair.

Fig. 1

Maps of repair fragments. (A) DHFR. The 6.2 kb XbaI fragment contains exons I and II. (B) V_HS107. The 4.8 kb XbaI fragment contains the V_HS107 gene segment (open box) rearranged to D and J_H1 segments. The fragment also contains J_H2, J_H3 and J_H4 gene segments. Locations of probes for transcription analyses are shown. Transcription start sites are indicated by bent arrows.

Fig. 2

Nuclear run-on analysis of RNA in two hybridoma lines. (A) ³²P-labeled nascent RNA transcripts from nuclei were hybridized to membranes containing the DHFR or V_HS107 probes depicted in Fig. 1. Two experiments for HPCG10 and HPCG17 cells are shown. (B) Hybridization intensity was normalized to equal nanomoles of each probe.

Fig. 3

Autoradiograms and analysis of DNA repair of the DHFR and V_HS107 genes. (A) Repair in HPCG10 cells. XbaI-digested DNA from different time points was treated with and without T4 denV endonuclease. A representative Southern blot is shown; size in kb is shown at the left of the blots. The blot was first hybridized to the DHFR probe, and then stripped and re-hybridized to a J_H2 probe. Data was obtained from densitometric scans, which were calculated in Table 1, and graphed as the average value of two separate biological experiments. (B) Repair in HPCG17 cells. Blots were prepared, hybridized and scanned as above. The results from three separate experiments are graphed.