Abstract
Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38 -/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38 -/- cells did not show a significant sensitivity to either UV or H 2 O 2 , a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38 -/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38 -/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.
| Original language | English |
|---|---|
| Article number | e0213383 |
| Journal | PloS one |
| Volume | 14 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - 2019 Mar 1 |
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ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Agricultural and Biological Sciences(all)
Cite this
PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching. / Tsuda, Masataka; Ogawa, Saki; Ooka, Masato; Kobayashi, Kaori; Hirota, Kouji; Wakasugi, Mitsuo; Matsunaga, Tsukasa; Sakuma, Tetsushi; Yamamoto, Takashi; Chikuma, Shunsuke; Sasanuma, Hiroyuki; Debatisse, Michelle; Doherty, Aidan J.; Fuchs, Robert P.; Takeda, Shunichi.
In: PloS one, Vol. 14, No. 3, e0213383, 01.03.2019.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching
AU - Tsuda, Masataka
AU - Ogawa, Saki
AU - Ooka, Masato
AU - Kobayashi, Kaori
AU - Hirota, Kouji
AU - Wakasugi, Mitsuo
AU - Matsunaga, Tsukasa
AU - Sakuma, Tetsushi
AU - Yamamoto, Takashi
AU - Chikuma, Shunsuke
AU - Sasanuma, Hiroyuki
AU - Debatisse, Michelle
AU - Doherty, Aidan J.
AU - Fuchs, Robert P.
AU - Takeda, Shunichi
PY - 2019/3/1
Y1 - 2019/3/1
N2 - Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38 -/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38 -/- cells did not show a significant sensitivity to either UV or H 2 O 2 , a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38 -/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38 -/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.
AB - Replicative DNA polymerases are frequently stalled at damaged template strands. Stalled replication forks are restored by the DNA damage tolerance (DDT) pathways, error-prone translesion DNA synthesis (TLS) to cope with excessive DNA damage, and error-free template switching (TS) by homologous DNA recombination. PDIP38 (Pol-delta interacting protein of 38 kDa), also called Pol δ-interacting protein 2 (PolDIP2), physically associates with TLS DNA polymerases, polymerase η (Polη), Polλ, and PrimPol, and activates them in vitro. It remains unclear whether PDIP38 promotes TLS in vivo, since no method allows for measuring individual TLS events in mammalian cells. We disrupted the PDIP38 gene, generating PDIP38 -/- cells from the chicken DT40 and human TK6 B cell lines. These PDIP38 -/- cells did not show a significant sensitivity to either UV or H 2 O 2 , a phenotype not seen in any TLS-polymerase-deficient DT40 or TK6 mutants. DT40 provides a unique opportunity of examining individual TLS and TS events by the nucleotide sequence analysis of the immunoglobulin variable (Ig V) gene as the cells continuously diversify Ig V by TLS (non-templated Ig V hypermutation) and TS (Ig gene conversion) during in vitro culture. PDIP38 -/- cells showed a shift in Ig V diversification from TLS to TS. We measured the relative usage of TLS and TS in TK6 cells at a chemically synthesized UV damage (CPD) integrated into genomic DNA. The loss of PDIP38 also caused an increase in the relative usage of TS. The number of UV-induced sister chromatid exchanges, TS events associated with crossover, was increased a few times in PDIP38 -/- human and chicken cells. Collectively, the loss of PDIP38 consistently causes a shift in DDT from TLS to TS without enhancing cellular sensitivity to DNA damage. We propose that PDIP38 controls the relative usage of TLS and TS increasing usage of TLS without changing the overall capability of DDT.
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U2 - 10.1371/journal.pone.0213383
DO - 10.1371/journal.pone.0213383
M3 - Article
VL - 14
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 3
M1 - e0213383
ER -