Analysis of V(D)J recombination in NHEJ-defective human cells & a structure:function analysis of mammalian Ku86.
2009-05
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Analysis of V(D)J recombination in NHEJ-defective human cells & a structure:function analysis of mammalian Ku86.
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2009-05
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Abstract
V(D)J recombination is a site-specific DNA recombination process required for
the assembly of immunoglobulin (Ig) and T cell receptor (TCR) genes, which is
accomplished through the use of DNA double strand breaks (Bassing et al., 2002). The
resulting hairpinned coding ends and the signal ends are rejoined primarily by the
canonical non-homologous end joining (C-NHEJ) pathway which consists of at least
seven factors: DNA-PKcs, Ku70, Ku86, Artemis, LigIV, XLF, XRCC4 (Rooney et al.,
2004). Deficiency of any these NHEJ factors leads to a phenotype of ‘Severe
Combined Immune Deficiency’ (SCID), which is characterized by a profound immune
dysfunction resulting from aberrant V(D)J recombination (Bassing et al., 2002; Rooney
et al., 2004; Zha et al., 2007). The accompanying blockage in B and T cell
development, increased ionizing radiation sensitivity, and developmental defects can all
be explained by the absence of the C-NHEJ pathway (Bassing et al., 2002; Rooney et
al., 2004; Zha et al., 2007).
Using rAAV-mediated gene targeting (Kohli et al., 2004), our laboratory has
generated loss-of-function mutations for all of the genes involved in NHEJ with the
exception of Artemis and XRCC4 (in press or manuscripts in preparation). We have
characterized these human cell lines that are either heterozygously or homozygously
defective for one of the NHEJ factors, to assess the role of each factor in V(D)J
recombination with three episomal substrates; pGG49 (signal joint), pGG51 (coding
joint) and pGG52 (inversion construct). We have found that there is a dramatic defect
in the frequency of V(D)J recombination in all null cell lines with all three substrates.
However, there is variation in the recombination frequency and the sequences utilized
at joints depending on which substrate and which cell line is used. This implies a
different role for each of the NHEJ factors in signal, coding or hybrid joint formation in
V(D)J recombination. We also observed similar recombination efficiency and
sequence variation patterns at the joint junction in wild type and all heterozygous cell
lines suggesting that residual NHEJ activity is enough for V(D)J recombination. This
contrasts with the role of these same genes in generalized DNA double-strand break
(DSB) repair for which a haploinsufficiency is often observed (Fattah et al., 2008b;
Ruis et al., 2008).
In chapter 2, we pursued the idea suggested by our work in chapter 1 that CNHEJ
factors may have different functions in different pathways. To this end, we
investigated a separation-of-function series of mutations for Ku86. For example, the
Ku heterodimer not only recognizes the broken ends of DNA at V(D)J recombinationmediated
DSBs and promotes their reassembly but also protects telomeres from
undergoing fusions. How Ku performs these seemingly opposite functions on different,
but structurally similar, substrates is unknown. As a precedent for the successful
application of this experimental approach, however, separation-of-function mutations
for Ku have been identified in yeast Ku (Bertuch and Lundblad, 2003; Palmbos et al.,
2005; Ribes-Zamora et al., 2007; Roy et al., 2004; Stellwagen et al., 2003).
Previously, several Ku86 mutant cell lines (sxi-1-4) derived from Golden Syrian
hamster V79-4 cells were isolated by forward and molecular genetic screens (He et al.,
1996b; Zdzienicka, 1995). All of these cell lines shared common features including
elevated sensitivity to radiation, defective DNA DSB repair, and aberrant V(D)J
recombination. By functionally complementing these mutant cell lines with wild-type
Ku86 cDNAs, it was determined that the absence of Ku86 caused these aberrant
phenotypes (Errami et al., 1996; Smider et al., 1994).
Here, we used the Ku86-defective sxi-3 cell line to determine if separation-offunction
of Ku86 in terms of DNA DSB repair, recombination and telomere
maintenance is conserved in mammalian cells. We introduced six point mutations
associated with defective telomere maintenance into the hamster Ku86 cDNA. These
cDNAs were then stably introduced into sxi-3 cells and various DNA repair and V(D)J
recombination assays were used to interrogate the lines. Domain-specific functions of
Ku86 were unveiled by these experiments.
In summary, we have used mutant human cell lines and mutant cDNAs to
elaborate the role of C-NHEJ factors — in general and Ku86 specifically — in a
plethora of DNA metabolic reactions. Our results have improved our understanding of
C-NHEJ reactions in humans.
Description
University of Minnesota Ph.D. dissertation. May 2009. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Dr. Eric A. Hendrickson. 1 computer file (PDF); viii 150 pages. Ill. (some col.)
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Kweon, Junghun. (2009). Analysis of V(D)J recombination in NHEJ-defective human cells & a structure:function analysis of mammalian Ku86.. Retrieved from the University Digital Conservancy, https://hdl.handle.net/11299/52233.
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