Tài liệu Báo cáo khoa học: Aggregative organization enhances the DNA end-joining process that is

Aggregative organization enhances the DNA end-joining

process that is mediated by DNA-dependent protein kinase

Masahiko Takahagi and Kouichi Tatsumi

Research Center for Radiation Safety, National Institute of Radiological Sciences, Chiba, Japan

DNA double-strand breaks (DSBs) are a serious threat

to the genetic integrity of organisms, causing cell death

if not repaired. The repair mechanism for DSBs resides

not only in catalytic processes but also in the associ￾ation with chromatin structures [1,2], although the

details in the higher-order context remain obscure.

Some evidence has implicated structural alterations in

the vicinity of DSB sites. DSBs can form nuclear foci

linked to phosphorylated histone H2AX (c-H2AX) [3],

which is responsible for the redistribution of repair

factors to DSB sites [4], although it is dispensable

for initial damage recognition [5]. Approximately 2000

c-H2AX molecules accumulate per focus in a normal

human cell, suggesting reorganization of chromosomal

DNA over a region of Mbp order [6]. c-H2AX-associ￾ated foci are morphologically dynamic; the DSB-con￾taining chromosome domains can be mobile, and in a

subpopulation of damaged cells, they can juxtapose

via an adhesion process irrespective of DNA repair

processes [7].

In addition to these large-scale responses to DSBs,

real-time analysis of the temporo-spatial distribution

of DNA repair factors in situ in living cells has been

providing us with striking information. For instance,

even following exposure to ionizing radiation (IR), the

DNA end-binding factor Ku moves rapidly throughout

the nucleus but associates transiently with filamentous

nuclear substrates [8]. A checkpoint regulator NBS1,

the product of the Nijmegen breakage syndrome gene,

shuttles rapidly between DSB sites and the flanking

chromatin [9]. These findings indicate that the action

of DSB-interactive proteins within the nuclear micro￾environment must be coupled with the mobile state of

those proteins.

Keywords

coacervate; DNA aggregation; DNA end￾joining; DNA-PK; S ⁄ MAR

Correspondence

K. Tatsumi, Research Center for Radiation

Safety, National Institute of Radiological

Sciences, 9–1, Anagawa 4, Inage-ku,

Chiba 263–8555, Japan

Fax: +81 43 255 6497

Tel: +81 43 206 3087

E-mail: [email protected]

(Received 7 April 2006, accepted 11 May

2006)

doi:10.1111/j.1742-4658.2006.05317.x

The occurrence of DNA double-strand breaks in the nucleus provokes in

its structural organization a large-scale alteration whose molecular basis is

still mostly unclear. Here, we show that double-strand breaks trigger pref￾erential assembly of nucleoproteins in human cellular fractions and that

they mediate the separation of large protein–DNA aggregates from aque￾ous solution. The interaction among the aggregative nucleoproteins pre￾sents a dynamic condition that allows the effective interaction of

nucleoproteins with external molecules like free ATP and facilitates intrin￾sic DNA end-joining activity. This aggregative organization is functionally

coacervate-like. The key component is DNA-dependent protein kinase

(DNA-PK), which can be characterized as a DNA-specific aggregation fac￾tor as well as a nuclear scaffold ⁄ matrix-interactive factor. In the context of

aggregation, the kinase activity of DNA-PK is essential for efficient DNA

end-joining. The massive and functional concentration of nucleoproteins

on DNA in vitro may represent a possible status of nuclear dynamics

in vivo, which probably includes the DNA-PK-dependent response to mul￾tiple double-strand breaks.

Abbreviations

DSB, double-strand break; DNA-PK, DNA-dependent protein kinase; EJ, end-joining; c-H2AX, phosphorylated histone H2AX; HMGB, high

mobility group box; IR, ionizing radiation; LD, linear duplex; NC, nicked circular; NF, nuclear fraction; SC, supercoiled; S ⁄ MAR,

scaffold ⁄ matrix attached region; ss, single strand; SSC, single-strand circular.

FEBS Journal 273 (2006) 3063–3075 ª 2006 The Authors Journal compilation ª 2006 FEBS 3063