Human DNA polymerase η activity and translocation is regulated by phosphorylation

Yih Wen Chen, James E. Cleaver, Zafer Hatahet, Richard E. Honkanen, Jang Yang Chang, Yun Yen, Kai Ming Chou

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

Human DNA polymerase η (pol η) can replicate across UV-induced pyrimidine dimers, and defects in the gene encoding pol η result in a syndrome called xeroderma pigmentosum variant (XP-V). XP-V patients are prone to the development of cancer in sun-exposed areas, and cells derived from XP-V patients demonstrate increased sensitivity to UV radiation and a higher mutation rate compared with wild-type cells. pol η has been shown to replicate across a wide spectrum of DNA lesions introduced by environmental or chemotherapeutic agents, or during nucleotide starvation, suggesting that the biological roles for pol η are not limited to repair of UV-damaged DNA. The high error rate of pol η requires that its intracellular activity be tightly regulated. Here, we show that the phosphorylation of pol η increased after UV irradiation, and that treatment with caffeine, siRNA against ATR, or an inhibitor of PKC (calphostin C), reduced the accumulation of pol η at stalled replication forks after UV irradiation or treatment with cisplatin and gemcitabine. Site-specific mutagenesis (S587A and T617A) of pol η at two putative PKC phosphorylation sites located in the protein-protein interaction domain prevented nuclear foci formation induced by UV irradiation or treatment with gemcitabine/cisplatin. In addition, XP-V cell lines stably expressing either the S587A or T617A mutant form of pol η were more sensitive to UV radiation and gemcitabine/cisplatin than control cells expressing wild-type pol η. These results suggest that phosphorylation is one mechanism by which the cellular activity of pol η is regulated.

Original languageEnglish
Pages (from-to)16578-16583
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume105
Issue number43
DOIs
Publication statusPublished - Oct 28 2008
Externally publishedYes

Fingerprint

gemcitabine
DNA-Directed DNA Polymerase
Phosphorylation
Cisplatin
Protein Interaction Domains and Motifs
Radiation
Pyrimidine Dimers
DNA
Solar System
Mutation Rate
Starvation
Site-Directed Mutagenesis
Caffeine
Small Interfering RNA
Therapeutics
Nucleotides
Cell Line
Variant type Xeroderma pigmentosum
Genes
Neoplasms

Keywords

  • Lesion bypass
  • Xeroderma pigmentosum variant

ASJC Scopus subject areas

  • General

Cite this

Human DNA polymerase η activity and translocation is regulated by phosphorylation. / Chen, Yih Wen; Cleaver, James E.; Hatahet, Zafer; Honkanen, Richard E.; Chang, Jang Yang; Yen, Yun; Chou, Kai Ming.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 43, 28.10.2008, p. 16578-16583.

Research output: Contribution to journalArticle

Chen, Yih Wen ; Cleaver, James E. ; Hatahet, Zafer ; Honkanen, Richard E. ; Chang, Jang Yang ; Yen, Yun ; Chou, Kai Ming. / Human DNA polymerase η activity and translocation is regulated by phosphorylation. In: Proceedings of the National Academy of Sciences of the United States of America. 2008 ; Vol. 105, No. 43. pp. 16578-16583.
@article{b797dc4ad0504c3fb5b93a9b151a145d,
title = "Human DNA polymerase η activity and translocation is regulated by phosphorylation",
abstract = "Human DNA polymerase η (pol η) can replicate across UV-induced pyrimidine dimers, and defects in the gene encoding pol η result in a syndrome called xeroderma pigmentosum variant (XP-V). XP-V patients are prone to the development of cancer in sun-exposed areas, and cells derived from XP-V patients demonstrate increased sensitivity to UV radiation and a higher mutation rate compared with wild-type cells. pol η has been shown to replicate across a wide spectrum of DNA lesions introduced by environmental or chemotherapeutic agents, or during nucleotide starvation, suggesting that the biological roles for pol η are not limited to repair of UV-damaged DNA. The high error rate of pol η requires that its intracellular activity be tightly regulated. Here, we show that the phosphorylation of pol η increased after UV irradiation, and that treatment with caffeine, siRNA against ATR, or an inhibitor of PKC (calphostin C), reduced the accumulation of pol η at stalled replication forks after UV irradiation or treatment with cisplatin and gemcitabine. Site-specific mutagenesis (S587A and T617A) of pol η at two putative PKC phosphorylation sites located in the protein-protein interaction domain prevented nuclear foci formation induced by UV irradiation or treatment with gemcitabine/cisplatin. In addition, XP-V cell lines stably expressing either the S587A or T617A mutant form of pol η were more sensitive to UV radiation and gemcitabine/cisplatin than control cells expressing wild-type pol η. These results suggest that phosphorylation is one mechanism by which the cellular activity of pol η is regulated.",
keywords = "Lesion bypass, Xeroderma pigmentosum variant",
author = "Chen, {Yih Wen} and Cleaver, {James E.} and Zafer Hatahet and Honkanen, {Richard E.} and Chang, {Jang Yang} and Yun Yen and Chou, {Kai Ming}",
year = "2008",
month = "10",
day = "28",
doi = "10.1073/pnas.0808589105",
language = "English",
volume = "105",
pages = "16578--16583",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
publisher = "National Academy of Sciences",
number = "43",

}

TY - JOUR

T1 - Human DNA polymerase η activity and translocation is regulated by phosphorylation

AU - Chen, Yih Wen

AU - Cleaver, James E.

AU - Hatahet, Zafer

AU - Honkanen, Richard E.

AU - Chang, Jang Yang

AU - Yen, Yun

AU - Chou, Kai Ming

PY - 2008/10/28

Y1 - 2008/10/28

N2 - Human DNA polymerase η (pol η) can replicate across UV-induced pyrimidine dimers, and defects in the gene encoding pol η result in a syndrome called xeroderma pigmentosum variant (XP-V). XP-V patients are prone to the development of cancer in sun-exposed areas, and cells derived from XP-V patients demonstrate increased sensitivity to UV radiation and a higher mutation rate compared with wild-type cells. pol η has been shown to replicate across a wide spectrum of DNA lesions introduced by environmental or chemotherapeutic agents, or during nucleotide starvation, suggesting that the biological roles for pol η are not limited to repair of UV-damaged DNA. The high error rate of pol η requires that its intracellular activity be tightly regulated. Here, we show that the phosphorylation of pol η increased after UV irradiation, and that treatment with caffeine, siRNA against ATR, or an inhibitor of PKC (calphostin C), reduced the accumulation of pol η at stalled replication forks after UV irradiation or treatment with cisplatin and gemcitabine. Site-specific mutagenesis (S587A and T617A) of pol η at two putative PKC phosphorylation sites located in the protein-protein interaction domain prevented nuclear foci formation induced by UV irradiation or treatment with gemcitabine/cisplatin. In addition, XP-V cell lines stably expressing either the S587A or T617A mutant form of pol η were more sensitive to UV radiation and gemcitabine/cisplatin than control cells expressing wild-type pol η. These results suggest that phosphorylation is one mechanism by which the cellular activity of pol η is regulated.

AB - Human DNA polymerase η (pol η) can replicate across UV-induced pyrimidine dimers, and defects in the gene encoding pol η result in a syndrome called xeroderma pigmentosum variant (XP-V). XP-V patients are prone to the development of cancer in sun-exposed areas, and cells derived from XP-V patients demonstrate increased sensitivity to UV radiation and a higher mutation rate compared with wild-type cells. pol η has been shown to replicate across a wide spectrum of DNA lesions introduced by environmental or chemotherapeutic agents, or during nucleotide starvation, suggesting that the biological roles for pol η are not limited to repair of UV-damaged DNA. The high error rate of pol η requires that its intracellular activity be tightly regulated. Here, we show that the phosphorylation of pol η increased after UV irradiation, and that treatment with caffeine, siRNA against ATR, or an inhibitor of PKC (calphostin C), reduced the accumulation of pol η at stalled replication forks after UV irradiation or treatment with cisplatin and gemcitabine. Site-specific mutagenesis (S587A and T617A) of pol η at two putative PKC phosphorylation sites located in the protein-protein interaction domain prevented nuclear foci formation induced by UV irradiation or treatment with gemcitabine/cisplatin. In addition, XP-V cell lines stably expressing either the S587A or T617A mutant form of pol η were more sensitive to UV radiation and gemcitabine/cisplatin than control cells expressing wild-type pol η. These results suggest that phosphorylation is one mechanism by which the cellular activity of pol η is regulated.

KW - Lesion bypass

KW - Xeroderma pigmentosum variant

UR - http://www.scopus.com/inward/record.url?scp=55949125726&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=55949125726&partnerID=8YFLogxK

U2 - 10.1073/pnas.0808589105

DO - 10.1073/pnas.0808589105

M3 - Article

VL - 105

SP - 16578

EP - 16583

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 43

ER -