Pold4, the fourth subunit of replicative polymerase δ, suppresses gene conversion in the immunoglobulin-variable gene in avian DT40 cells

Warsaw breakage syndrome, a developmental disorder caused by mutations in the DDX11/ChlR1 helicase, shows cellular features of genome instability similar to Fanconi anemia (FA). Here we report that DDX11-deficient avian DT40 cells exhibit interstrand crosslink (ICL)-induced chromatid breakage, with DDX11 functioning as backup for the FA pathway in regard to ICL repair. Importantly, we establish that DDX11 acts jointly with the 9-1-1 checkpoint clamp and its loader, RAD17, primarily in a postreplicative fashion, to promote homologous recombination repair of bulky lesions, but is not required for intra-S checkpoint activation or efficient fork progression. Notably, we find that DDX11 also promotes diversification of the chicken Ig-variable gene, a process triggered by programmed abasic sites, by facilitating both hypermutation and homeologous recombination-mediated gene conversion. Altogether, our results uncover that DDX11 orchestrates jointly with 9-1-1 and its loader, RAD17, DNA damage tolerance at sites of bulky lesions, and endogenous abasic sites. These functions may explain the essential roles of DDX11 and its similarity with 9-1-1 during development.

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Role for RAD18 in Homologous Recombination in DT40 Cells

Molecular and Cellular Biology ◽

10.1128/mcb.01291-06 ◽

2006 ◽

Vol 26 (21) ◽

pp. 8032-8041 ◽

Cited By ~ 36

Author(s):

Dávid Szüts ◽

Laura J. Simpson ◽

Sarah Kabani ◽

Mitsuyoshi Yamazoe ◽

Julian E. Sale

Keyword(s):

Dna Damage ◽

Homologous Recombination ◽

Gene Conversion ◽

Early Stage ◽

Current Evidence ◽

Recombination Reaction ◽

Immunoglobulin Gene ◽

Postreplication Repair ◽

Strand Breaks ◽

Dt40 Cells

ABSTRACT RAD18 is an E3 ubiquitin ligase that catalyzes the monoubiquitination of PCNA, a modification central to DNA damage bypass and postreplication repair in both yeast and vertebrates. Although current evidence suggests that homologous recombination provides an essential backup in vertebrate rad18 mutants, we show that in chicken DT40 cells this is not the case and that RAD18 plays a role in the recombination reaction itself. Gene conversion tracts in the immunoglobulin locus of rad18 cells are shorter and are associated with an increased frequency of deletions and duplications. rad18 cells also exhibit reduced efficiency of gene conversion induced by targeted double-strand breaks in a reporter construct. Blocking an early stage of the recombination reaction by disruption of XRCC3 not only suppresses immunoglobulin gene conversion but also prevents the aberrant immunoglobulin gene rearrangements associated with RAD18 deficiency, reverses the elevated sister chromatid exchange of the rad18 mutant, and reduces its sensitivity to DNA damage. Together, these data suggest that homologous recombination is toxic in the absence of RAD18 and show that, in addition to its established role in postreplication repair, RAD18 is also required for the orderly completion of gene conversion.

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Similar Effects of Brca2 Truncation and Rad51 Paralog Deficiency on Immunoglobulin V Gene Diversification in DT40 Cells Support an Early Role for Rad51 Paralogs in Homologous Recombination

Molecular and Cellular Biology ◽

10.1128/mcb.25.3.1124-1134.2005 ◽

2005 ◽

Vol 25 (3) ◽

pp. 1124-1134 ◽

Cited By ~ 64

Author(s):

Atsushi Hatanaka ◽

Mitsuyoshi Yamazoe ◽

Julian E. Sale ◽

Minoru Takata ◽

Kazuhiko Yamamoto ◽

...

Keyword(s):

Gene Segment ◽

Dna Recombination ◽

Suppressor Gene ◽

Nucleotide Substitutions ◽

Gene Diversification ◽

Brca2 Protein ◽

Rad51 Paralogs ◽

Error Prone Repair ◽

Variable Gene ◽

Dt40 Cells

ABSTRACT BRCA2 is a tumor suppressor gene that is linked to hereditary breast and ovarian cancer. Although the Brca2 protein participates in homologous DNA recombination (HR), its precise role remains unclear. From chicken DT40 cells, we generated BRCA2 gene-deficient cells which harbor a truncation at the 3′ end of the BRC3 repeat (brca2tr). Comparison of the characteristics of brca2tr cells with those of other HR-deficient DT40 clones revealed marked similarities with rad51 paralog mutants (rad51b, rad51c, rad51d, xrcc2, or xrcc3 cells). The phenotypic similarities include a shift from HR-mediated diversification to single-nucleotide substitutions in the immunoglobulin variable gene segment and the partial reversion of this shift by overexpression of Rad51. Although recent evidence supports at least Xrcc3 and Rad51C playing a role late in HR, our data suggest that Brca2 and the Rad51 paralogs may also contribute to HR at the same early step, with their loss resulting in the stimulation of an alternative, error-prone repair pathway.

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SPARTAN promotes genetic diversification of the immunoglobulin-variable gene locus in avian DT40 cells

DNA Repair ◽

10.1016/j.dnarep.2018.06.003 ◽

2018 ◽

Vol 68 ◽

pp. 50-57 ◽

Cited By ~ 1

Author(s):

Arisa Nakazato ◽

Kinumi Kajita ◽

Masato Ooka ◽

Remi Akagawa ◽

Takuya Abe ◽

...

Keyword(s):

Gene Locus ◽

Variable Gene ◽

Dt40 Cells

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Immunoglobulin gene conversion in chicken DT40 cells largely proceeds through an abasic site intermediate generated by excision of the uracil produced by AID-mediated deoxycytidine deamination

European Journal of Immunology ◽

10.1002/eji.200324631 ◽

2004 ◽

Vol 34 (2) ◽

pp. 504-508 ◽

Cited By ~ 56

Author(s):

Javier M. Di Noia ◽

Michael S. Neuberger

Keyword(s):

Gene Conversion ◽

Immunoglobulin Gene ◽

Abasic Site ◽

Dt40 Cells

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The 9-1-1 DNA Clamp Is Required for Immunoglobulin Gene Conversion

Molecular and Cellular Biology ◽

10.1128/mcb.00156-08 ◽

2008 ◽

Vol 28 (19) ◽

pp. 6113-6122 ◽

Cited By ~ 20

Author(s):

Alihossein Saberi ◽

Makoto Nakahara ◽

Julian E. Sale ◽

Koji Kikuchi ◽

Hiroshi Arakawa ◽

...

Keyword(s):

Homologous Recombination ◽

Gene Conversion ◽

Translesion Synthesis ◽

Strand Break ◽

Double Strand Break ◽

Immunoglobulin Gene ◽

Abasic Sites ◽

Immunoglobulin Locus ◽

Activation Induced Deaminase ◽

Dt40 Cells

ABSTRACT Chicken DT40 cells deficient in the 9-1-1 checkpoint clamp exhibit hypersensitivity to a variety of DNA-damaging agents. Although recent work suggests that, in addition to its role in checkpoint activation, this complex may play a role in homologous recombination and translesion synthesis, the cause of this hypersensitivity has not been studied thoroughly. The immunoglobulin locus of DT40 cells allows monitoring of homologous recombination and translesion synthesis initiated by activation-induced deaminase (AID)-dependent abasic sites. We show that both the RAD9 −/− and RAD17 −/− mutants exhibit substantially reduced immunoglobulin gene conversion. However, the level of nontemplated immunoglobulin point mutation increased in these mutants, a finding that is reminiscent of the phenotype resulting from the loss of RAD51 paralogs or Brca2. This suggests that the 9-1-1 complex does not play a central role in translesion synthesis in this context. Despite reduced immunoglobulin gene conversion, the RAD9 −/− and RAD17 −/− cells do not exhibit a prominent defect in double-strand break-induced gene conversion or a sensitivity to camptothecin. This suggests that the roles of Rad9 and Rad17 may be confined to a subset of homologous recombination reactions initiated by replication-stalling lesions rather than those associated with double-strand break repair.

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