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V(D)J recombination in mammalian cell mutants defective in DNA double-strand break repair
F Pergola1, M Z Zdzienicka, M R Lieber
1Department of Pathology, Stanford University School of Medicine, California 94305-5324.
Abstract:
V(D)J recombination has been examined in several X-ray-sensitive and double-strand break repair-deficient Chinese hamster cell mutants. Signal joint formation was affected in four mutants (xrs 5, XR-1, V-3, and XR-V9B cells, representing complementation groups 1 through 4, respectively) defective in DNA double-strand break rejoining. Among these four, V-3 and XR-V9B were the most severely affected. Only in V-3 was coding joint formation also affected. Ataxia telangiectasia-like hamster cell mutants (V-E5 and V-G8), which are normal for double-strand break repair but are X ray sensitive, were normal for all aspects of the V(D)J recombination reaction, indicating that X-ray sensitivity is not the common denominator but that the deficiency in double-strand break repair appears to be. The abnormality at the signal joints consisted of an elevated incidence of nucleotide loss from each of the two signal ends. Interestingly, in complementation groups 1 (xrs 5) and 2 (XR-1), signal joint formation was within the normal range under some transfection conditions. This suggests that the affected gene products in these two complementation groups are not catalytic components. Instead, they may be either secondary or stochiometric components involved in the later stages of both the V(D)J recombination reaction and double-strand break repair. The fact that such factors can affect the precision of the signal joint has mechanistic implications for V(D)J recombination.
Insights
DNA double-strand break repair deficiency impairs V(D)J recombination signal joint formation in Chinese hamster cells. This suggests repair proteins are crucial for V(D)J recombination accuracy.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- V(D)J recombination is essential for adaptive immunity.
- DNA double-strand break (DSB) repair pathways are critical for genomic stability.
- X-ray sensitivity in cell mutants can indicate defects in DNA repair.
Purpose of the Study:
- To investigate the role of DNA double-strand break repair in V(D)J recombination.
- To identify specific defects in V(D)J recombination associated with DSB repair deficiencies.
Main Methods:
- Analysis of V(D)J recombination in X-ray-sensitive Chinese hamster cell mutants.
- Assessment of signal and coding joint formation in various mutant cell lines.
- Complementation analysis to group mutants based on defective genes.
Main Results:
- Four DSB repair-deficient mutants (xrs 5, XR-1, V-3, XR-V9B) showed impaired signal joint formation.
- V-3 and XR-V9B mutants exhibited the most severe defects; only V-3 was also affected in coding joint formation.
- Ataxia telangiectasia-like mutants, proficient in DSB repair, showed normal V(D)J recombination, highlighting DSB repair proficiency as key, not X-ray sensitivity.
- Abnormalities included increased nucleotide loss at signal joints.
- Mutants xrs 5 and XR-1 showed near-normal signal joint formation under certain conditions, suggesting non-catalytic roles for their affected gene products.
Conclusions:
- Deficiency in DNA double-strand break repair directly impacts V(D)J recombination, specifically signal joint formation.
- The affected gene products in some mutants may be secondary or stoichiometric factors involved in later stages of both DSB repair and V(D)J recombination.
- These findings have mechanistic implications for the precision of V(D)J recombination and its link to DNA repair.