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KMID : 0380020090240040403
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Korean Journal of Biotechnology and Bioengineering
2009 Volume.24 No. 4 p.403 ~ p.409
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H2AX Directly Interacts with BRCA1 and BARD1 via its NLS and BRCT Domain Respectively in vitro
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Bae Seung-Hee
Choe Tae-Boo
Lee Sun-Mi
Kim Su-Mi
Kim Cha-Soon
Seong Ki-Moon
Jin Young-Woo
An Sung-Kwan
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Abstract
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H2AX, a crucial component of chromatin, is implicated in DNA repair, cell cycle check point and tumor suppression. The aim of this study was to identify direct binding partners of H2AX to regulate cellular responses to above mechanisms. Literature reviews and bioinformatical tools were attempted intensively to find binding partners of H2AX, which resulted in identifying two potential proteins, breast cancer-1 (BRCA1) and BRCA1-associated RING domain 1 (BARD1). Although it has been reported in vivo that BRCA1 co-localizes with H2AX at the site of DNA damage, their biochemical mechanism for H2AX were however only known that the complex monoubiquitinates histone monomers, including unphosphorylated H2AX in vitro. Therefore, it is important to know whether the complex directly interacts with H2AX, and also which regions of these are specifically mediated for the interaction. Using in vitro GST pull-down assay, we present here that BRCA1 and BARD1 directly bind to H2AX. Moreover, through combinational approaches of domain analysis, fragment clonings and in vitro binding assay, we revealed molecular details of the BRCA1-H2AX and BARD1-H2AX complex. These data provide the potential evidence that each of the BRCA1 nuclear localization signal (NLS) and BARD1 BRCA1 C-terminal (BRCT) repeat domain is the novel mediator of H2AX recognition.
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KEYWORD
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H2AX, BRCA1, BARD1, interaction
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