SUMOylation of PCNA by PIAS1 and PIAS4 promotes template switch in the chicken and human B cell lines

Mohiuddin Mohiuddin; Terry John Evans; Md Maminur Rahman; Islam Shamima Keka; Masataka Tsuda; Hiroyuki Sasanuma; Shunichi Takeda

doi:10.1073/pnas.1716349115

SUMOylation of PCNA by PIAS1 and PIAS4 promotes template switch in the chicken and human B cell lines

Proc Natl Acad Sci U S A. 2018 Dec 11;115(50):12793-12798. doi: 10.1073/pnas.1716349115. Epub 2018 Nov 28.

Authors

Mohiuddin Mohiuddin¹, Terry John Evans¹, Md Maminur Rahman¹, Islam Shamima Keka¹, Masataka Tsuda¹, Hiroyuki Sasanuma¹, Shunichi Takeda²

Affiliations

¹ Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, 606-8501 Kyoto, Japan.
² Department of Radiation Genetics, Graduate School of Medicine, Kyoto University, 606-8501 Kyoto, Japan stakeda@rg.med.kyoto-u.ac.jp.

Abstract

DNA damage tolerance (DDT) releases replication blockage caused by damaged nucleotides on template strands employing two alternative pathways, error-prone translesion DNA synthesis (TLS) and error-free template switch (TS). Lys164 of proliferating cell nuclear antigen (PCNA) is SUMOylated during the physiological cell cycle. To explore the role for SUMOylation of PCNA in DDT, we characterized chicken DT40 and human TK6 B cells deficient in the PIAS1 and PIAS4 small ubiquitin-like modifier (SUMO) E3 ligases. DT40 cells have a unique advantage in the phenotypic analysis of DDT as they continuously diversify their immunoglobulin (Ig) variable genes by TLS and TS [Ig gene conversion (GC)], both relieving replication blocks at abasic sites without accompanying by DNA breakage. Remarkably, PIAS1^-/-/PIAS4^-/- cells displayed a multifold decrease in SUMOylation of PCNA at Lys164 and over a 90% decrease in the rate of TS. Likewise, PIAS1^-/-/PIAS4^-/- TK6 cells showed a shift of DDT from TS to TLS at a chemosynthetic UV lesion inserted into the genomic DNA. The PCNA^K164R/K164R mutation caused a ∼90% decrease in the rate of Ig GC and no additional impact on PIAS1^-/-/PIAS4^-/- cells. This epistatic relationship between the PCNA^K164R/K164R and the PIAS1^-/-/PIAS4^-/- mutations suggests that PIAS1 and PIAS4 promote TS mainly through SUMOylation of PCNA at Lys164. This idea is further supported by the data that overexpression of a PCNA-SUMO1 chimeric protein restores defects in TS in PIAS1^-/-/PIAS4^-/- cells. In conclusion, SUMOylation of PCNA at Lys164 promoted by PIAS1 and PIAS4 ensures the error-free release of replication blockage during physiological DNA replication in metazoan cells.

Keywords: PCNA; PIAS1; PIAS4; SUMOylation; template switching (TS).

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
B-Lymphocytes / metabolism*
Cell Cycle / genetics
Cell Line
Chickens / genetics*
DNA Damage / genetics
DNA Repair / genetics
DNA Replication / genetics
Gene Conversion / genetics
Genes, Immunoglobulin / genetics
Humans
Immunoglobulin Variable Region / genetics
Poly-ADP-Ribose Binding Proteins / genetics*
Proliferating Cell Nuclear Antigen / genetics*
Protein Inhibitors of Activated STAT / genetics*
Sumoylation / genetics*
Ubiquitin-Protein Ligases / genetics

Substances

Immunoglobulin Variable Region
Poly-ADP-Ribose Binding Proteins
Proliferating Cell Nuclear Antigen
Protein Inhibitors of Activated STAT
Ubiquitin-Protein Ligases