| Project/Area Number |
09680592
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Okayama University |
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Principal Investigator |
TSUTSUKI Ken Okayama University, Medical School, Assistant Professor, 医学部, 助教授 (70108158)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1999: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1998: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1997: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | DNA binding protein / AT-tracts / Nuclear matrix / Nuclear scaffold / MAR / SAR |
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| Research Abstract |
SP120 is a major nuclear matrix protein that binds specifically to the matrix attachment region DNA (MAR) to organize the genome into domains. To delineate sequence requirements for the recognition by SP120, we used synthetic concatemers as an artificial MAR. The protein showed a high affinity to the concatemer synthesized by ligation of a duplex monomer (26 nt) with 4-bp overhangs. The monomer duplex contained two short homopolymeric AT-tracts although the overall AT content was only 42%. Orientation of the monomer units in the concatemer is randomized because of the palindromic overhang and this type of concatemer was referred to as "RC" for random concatemer. Other types of concatemers ("DC" and "AC" representing direct and alternating orientations, respectively) were also synthesized by manipulating the overhang sequences. Comparison of affinities of these concatemers to purified SP120, using a filter binding assay or southwestern blotting, gave an unequivocal result in that only RC showed a significant binding. The binding was abolished effectively by poly(dA). poly(dT) but not by poly(dG).poly(dC), suggesting that the AT-tracts in the RC are the recognition signal for protein binding. It should be emphasized that all the concatemers have identical AT content and the same number of AT-tracts. Thus, the result strongly suggests that the binding activity is accounted for by only a subset of RC molecules with a particular pattern of AT-tracts distribution that are contained in the heterogeneous population. The possible involvement of AT-tracts in the protein recognition was tested more directly with RCs possessing altered AT-tracts. When both AT-tracts were disrupted by replacing T's with G/C or A, all the mutant concatemers showed a marginal binding to SP120, indicating that the intactness of AT-tracts are indeed required for the binding.
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