PDIP38/PolDIP2 controls the DNA damage tolerance pathways by increasing the relative usage of translesion DNA synthesis over template switching.

Masataka Tsuda,Aidan J Doherty,Tetsushi Sakuma,Saki Ogawa,Michelle Debatisse,Shunsuke Chikuma,Masato Ooka,Tsukasa Matsunaga,Mitsuo Wakasugi,Hiroyuki Sasanuma,Robert P Fuchs,Kaori Kobayashi,Shunichi Takeda,Takashi Yamamoto,Kouji Hirota

doi:10.1371/journal.pone.0213383

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 H2O2, 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.

Highlights

DNA replication is a fragile system and is frequently stalled at damaged template strands
The S-phase cells account for 70% and 50% of the whole cell cycle time of DT40 and TK6 cells, respectively, which high percentages of S-phase cells allow for sensitive detection of defects in DNA damage tolerance (DDT) pathways functioning during DNA replication
The PDIP38-/- clones derived from DT40 and TK6 proliferated with normal kinetics (S3D and S4D Figs)

Summary

Introduction

DNA replication is a fragile system and is frequently stalled at damaged template strands. Stalled replication forks are released by the two major DDT pathways, TLS and TS. Homologous recombination (HR) facilitates transient switching of replication primers from the damaged template strand to the newly synthesized sister chromatid via a mechanism that remains very poorly understood even in Saccharomyces cerevisiae (S. cerevisiae) [3,4,5]. Proliferating Cell Nuclear Antigen (PCNA), a molecular sliding clamp for replicative DNA polymerase, plays the central role in a molecular switch from replicative polymerases to TLS polymerases [9]. In response to replication blockage, the mono-ubiquitination of PCNA by Rad ubiquitylation enzyme facilitates the recruitment of TLS polymerases to stalled replication forks [10]. Rev facilitates TLS by associating with several TLS polymerases such as Polη, Polκ, Polι, Polz [11]

Methods

Results

Conclusion