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
N1 - Funding Information:
This work was supported by Biotechnology and Biological Sciences Research Council BB/H019723/1 Dr. Aidan J Doherty, Biotechnology and Biological Sciences Research Council BB/M008800/1 Dr. Aidan J Doherty, JSPS KAKENHI Grant Number JP16H06306 Dr. Shunichi Takeda, JSPS Core-to-Core Program, A. Advanced Research Networks Dr. Shunichi Takeda, JSPS Core-to-Core Program Dr. Shunichi Takeda. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Multilabel Plate Reader ARVO X5 was performed at the Medical Research Support Center, Graduation School of Medicine, Kyoto University. We acknowledge the Radioisotope Research Center in Kyoto University for support in the use of isotopes. The authors thank R. Tanaka, K. Iwamoto, M. Kitaoka, M. Kato, and A. Kobayashi for technical assistant and the members of the Department of Radiation Genetics for their comments.
PY - 2019/3
Y1 - 2019/3
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
C2 - 30840704
AN - SCOPUS:85062641443
VL - 14
JO - PLoS One
JF - PLoS One
SN - 1932-6203
IS - 3
M1 - e0213383
ER -