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J. Biol. Chem., Vol. 279, Issue 30, 31164-31170, July 23, 2004

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Human SAD1 Kinase Is Involved in UV-induced DNA Damage Checkpoint Function^*

Rui Lu, Hiroyuki Niida, and Makoto Nakanishi

From the Department of Biochemistry and Cell Biology, Graduate School of Medical Sciences, Nagoya City University, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan

Checkpoint activation by DNA damage during G₂ prevents activation of cyclin B/Cdc2 complexes, and as a consequence, mitotic entry is blocked. Although initiation and maintenance of G₂ arrest are known to be regulated by at least two distinct signaling pathways, including those of p38MAPK and ataxia-telangiectasia-mutated (ATM)- and Rad3-related (ATR)-Chk1 in higher eukaryotes, the actual number of signaling pathways involved in this regulation is still elusive. In the present study, we identified human SAD1 (hsSAD1) by searching a sequence data base. The predicted hsSAD1 protein comprises 778 amino acids and shares significant homology with the fission yeast Cdr2, a mitosis-regulatory kinase, and Caenorhabditis elegans SAD1, a neuronal cell polarity regulator. HsSAD1 transcript was expressed ubiquitously with the highest levels of expression in brain and testis. HsSAD1 specifically phosphorylated Wee1A, Cdc25-C, and -B on Ser-642, Ser-216, and Ser-361 in vitro, respectively. Overexpression of hsSAD1 resulted in an increased phosphorylation of Cdc25C on Ser-216 in vivo. DNA damage induced by UV or methyl methane sulfonate but not by IR enhanced endogenous hsSAD1 kinase activity in a caffeine-sensitive manner and caused translocation of its protein from cytoplasm to nucleus. Overexpression of wild-type hsSAD1 induced G₂/M arrest in HeLa S2 cells. Furthermore, UV-induced G₂/M arrest was partially abrogated by the reduced expression of hsSAD1 using small interfering RNA. These results suggest that hsSAD1 acts as checkpoint kinase upon DNA damage induced by UV or methyl methane sulfonate. The identification of this new kinase suggests the existence of an alternative checkpoint pathway other than those of ATR-Chk1 and p38MAPK.

Received for publication, April 28, 2004 , and in revised form, May 18, 2004.

^* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan (to M. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-52-853-8144; Fax: 81-52-842-3955; E-mail: mkt-naka{at}med.nagoya-cu.ac.jp.

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				L. Wu, K. Luo, Z. Lou, and J. Chen MDC1 regulates intra-S-phase checkpoint by targeting NBS1 to DNA double-strand breaks PNAS, August 12, 2008; 105(32): 11200 - 11205. [Abstract] [Full Text] [PDF]

				H. Niida and M. Nakanishi DNA damage checkpoints in mammals Mutagenesis, January 1, 2006; 21(1): 3 - 9. [Abstract] [Full Text] [PDF]