Faculty Profiles - MASUTANI, Chikahide

Sphingosine: A Modulator of Human Translesion DNA Polymerase Activity. Reviewed

Kamath-Loeb AS, Balakrishna S, Whittington D, Shen JC, Emond MJ, Okabe T, Masutani C, Hanaoka F, Nishimura S, Loeb LA.

Journal of Biological Chemistry 2014.7

Guanine- 5-carboxylcytosine base pairs mimic mismatches during DNA replication. Reviewed International journal

Toshihiro Shibutani, Shinsuke Ito, Mariko Toda, Rie Kanao, Leonard B Collins, Marika Shibata, Miho Urabe, Haruhiko Koseki, Yuji Masuda, James A Swenberg, Chikahide Masutani, Fumio Hanaoka, Shigenori Iwai, Isao Kuraoka

Scientific reports Vol. 4 page： 5220 - 5220 2014.6

Language：Japanese Publishing type：Research paper (scientific journal)

The genetic information encoded in genomes must be faithfully replicated and transmitted to daughter cells. The recent discovery of consecutive DNA conversions by TET family proteins of 5-methylcytosine into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC) suggests these modified cytosines act as DNA lesions, which could threaten genome integrity. Here, we have shown that although 5caC pairs with guanine during DNA replication in vitro, G·5caC pairs stimulated DNA polymerase exonuclease activity and were recognized by the mismatch repair (MMR) proteins. Knockdown of thymine DNA glycosylase increased 5caC in genome, affected cell proliferation via MMR, indicating MMR is a novel reader for 5caC. These results suggest the epigenetic modification products of 5caC behave as DNA lesions.

DOI： 10.1038/srep05220

A cyclobutane thymine-N4-methylcytosine dimer is resistant to hydrolysis but strongly blocks DNA synthesis. Reviewed International journal

Junpei Yamamoto, Tomoko Oyama, Tomohiro Kunishi, Chikahide Masutani, Fumio Hanaoka, Shigenori Iwai

Nucleic acids research Vol. 42 ( 3 ) page： 2075 - 2084 2014.2

Language：Japanese Publishing type：Research paper (scientific journal)

Exposure of DNA to ultraviolet light produces harmful crosslinks between adjacent pyrimidine bases, to form cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts. The CPD is frequently formed, and its repair mechanisms have been exclusively studied by using a CPD formed at a TT site. On the other hand, biochemical analyses using CPDs formed within cytosine-containing sequence contexts are practically difficult, because saturated cytosine easily undergoes hydrolytic deamination. Here, we found that N-alkylation of the exocyclic amino group of 2'-deoxycytidine prevents hydrolysis in CPD formation, and an N-methylated cytosine-containing CPD was stable enough to be derivatized into its phosphoramidite building block and incorporated into oligonucleotides. Kinetic studies of the CPD-containing oligonucleotide indicated that its lifetime under physiological conditions is relatively long (∼ 7 days). In biochemical analyses using human DNA polymerase η, incorporation of TMP opposite the N-methylcytosine moiety of the CPD was clearly detected, in addition to dGMP incorporation, and the incorrect TMP incorporation blocked DNA synthesis. The thermodynamic parameters confirmed the formation of this unusual base pair.

DOI： 10.1093/nar/gkt1039

En bloc transfer of polyubiquitin chains to PCNA in vitro is mediated by two different human E2-E3 pairs

Interactions between PCNA and DNA polymerases of Y-family

Masuda Yuji, Kanao Rie, Masutani Chikahide

GENES & GENETIC SYSTEMS Vol. 88 ( 6 ) page： 360 - 360 2013.12

Novel mechanisms of PCNA ubiquitination

Masuda Yuji, Masutani Chikahide

GENES & GENETIC SYSTEMS Vol. 87 ( 6 ) page： 387 - 387 2012.12

En bloc transfer of poly-ubiquitin chains to PCNA in vitro is mediated by two human E2-E3 pairs. Reviewed

*Masuda, Y., Suzuki, M., Kawai, H., Hishiki, A., Hashimoto, H., Masutani, C., Hishida, T., Suzuki, F., *Kamiya. K.

Nucl. Acids Res. Vol. 40 ( 20 ) page： 10394-10407 2012.11

Masuda Yuji, Suzuki Miki, Kawai Hidehiko, Hishiki Asami, Hashimoto Hiroshi, Masutani Chikahide, Hishida Takashi, Suzuki Fumio, Kamiya Kenji

NUCLEIC ACIDS RESEARCH Vol. 40 ( 20 ) page： 10394 - 10407 2012.11

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Nucleic Acids Research

Post-replication DNA repair in eukaryotes is regulated by ubiquitination of proliferating cell nuclear antigen (PCNA). Monoubiquitination catalyzed by RAD6-RAD18 (an E2-E3 complex) stimulates translesion DNA synthesis, whereas polyubiquitination, promoted by additional factors such as MMS2-UBC13 (a UEV-E2 complex) and HLTF (an E3 ligase), leads to template switching in humans. Here, using an in vitro ubiquitination reaction system reconstituted with purified human proteins, we demonstrated that PCNA is polyubiquitinated predominantly via en bloc transfer of a pre-formed ubiquitin (Ub) chain rather than by extension of the Ub chain on monoubiquitinated PCNA. Our results support a model in which HLTF forms a thiol-linked Ub chain on UBC13 (UBC13∼Ubn) and then transfers the chain to RAD6∼Ub, forming RAD6∼Ubn+1. The resultant Ub chain is subsequently transferred to PCNA by RAD18. Thus, template switching may be promoted under certain circumstances in which both RAD18 and HLTF are coordinately recruited to sites of stalled replication. © 2012 The Author(s).

DOI： 10.1093/nar/gks763

Human DNA Polymerase η and Its Regulatory Mechanisms

Masutani Chikahide

Genes and environment : the official journal of the Japanese Environmental Mutagen Society Vol. 34 ( 2 ) page： 63 - 69 2012.5

Language：English Publishing type：Research paper (scientific journal) Publisher：日本環境変異原学会

Defects in DNA polymerase (Pol) η result in a cancer-prone and UV-sensitive inherited syndrome, a variant form of xeroderma pigmentosum, suggesting that Polη plays a vital role in preventing UV-induced skin cancers. In fact, Polη can catalyze translesion synthesis (TLS) past prominent UV-induced lesions efficiently and accurately. However, Polη is intrinsically an error-prone DNA polymerase, like other TLS polymerases. Biochemical, structural and physiological studies revealed that Polη and other TLS polymerases participate in multiple mutagenic mechanisms, including somatic hypermutation of immunoglobulin genes. Protein-protein interactions between Polη and PCNA, TLS polymerases, RAD18 and DNA repair proteins, as well as their posttranslational modifications, have been shown to be important for regulating Polη.<br>

DOI： 10.3123/jemsge.34.63

NBS1 Recruits RAD18 via a RAD6-like Domain and Regulates Pol eta-Dependent Translesion DNA Synthesis

Yanagihara Hiromi, Kobayashi Junya, Tateishi Satoshi, Kato Akihiro, Matsuura Shinya, Tauchi Hiroshi, Yamada Kouichi, Takezawa Jun, Sugasawa Kaoru, Masutani Chikahide, Hanaoka Fumio, Weemaes Corry M., Mori Toshio, Zou Lee, Komatsu Kenshi

MOLECULAR CELL Vol. 43 ( 5 ) page： 788 - 797 2011.9

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Molecular Cell

Translesion DNA synthesis, a process orchestrated by monoubiquitinated PCNA, is critical for DNA damage tolerance. While the ubiquitin-conjugating enzyme RAD6 and ubiquitin ligase RAD18 are known to monoubiquitinate PCNA, how they are regulated by DNA damage is not fully understood. We show that NBS1 (mutated in Nijmegen breakage syndrome) binds to RAD18 after UV irradiation and mediates the recruitment of RAD18 to sites of DNA damage. Disruption of NBS1 abolished RAD18-dependent PCNA ubiquitination and Polη focus formation, leading to elevated UV sensitivity and mutation. Unexpectedly, the RAD18-interacting domain of NBS1, which was mapped to its C terminus, shares structural and functional similarity with the RAD18-interacting domain of RAD6. These domains of NBS1 and RAD6 allow the two proteins to interact with RAD18 homodimers simultaneously and are crucial for Polη-dependent UV tolerance. Thus, in addition to chromosomal break repair, NBS1 plays a key role in translesion DNA synthesis. © 2011 Elsevier Inc.

DOI： 10.1016/j.molcel.2011.07.026