Malkov V A, Camerini-Otero R D
Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1810, USA.
J Biol Chem. 1995 Dec 15;270(50):30230-3. doi: 10.1074/jbc.270.50.30230.
To function as a repair and recombination protein, RecA has to be assembled as an active filament on single-stranded DNA in the presence of ATP or its analogs. We have identified amino acids in the primary DNA binding site of RecA that interact with single-stranded DNA by photocross-linking. A nucleoprotein complex consisting of RecA protein bound to a monosubstituted oligonucleotide bearing a 5-iododeoxyuracil cross-linking moiety was irradiated with long wavelength ultraviolet radiation to effect cross-linking with RecA protein. Subsequent trypsin digestion, followed by purification and peptide sequencing, revealed the cross-linking of two independent peptides, amino acid residues 153-169 and 199-216. Met164 from loop L1 and Phe203 from loop L2 were determined to be the exact points of cross-linking. Thus, our data confirm and extend predictions about the DNA binding domain of RecA protein based on the molecular structure of RecA (Story, R. M., Weber, I. T., and Steitz, T. A. (1992) Nature 355, 318-325).
为了发挥修复和重组蛋白的功能，RecA必须在ATP或其类似物存在的情况下，在单链DNA上组装成活性细丝。我们已经通过光交联鉴定出RecA主要DNA结合位点中与单链DNA相互作用的氨基酸。用长波长紫外线照射由RecA蛋白与带有5-碘脱氧尿嘧啶交联部分的单取代寡核苷酸结合形成的核蛋白复合物，以实现与RecA蛋白的交联。随后进行胰蛋白酶消化，然后纯化和肽测序，揭示了两个独立肽段（氨基酸残基153 - 169和199 - 216）的交联。确定来自环L1的Met164和来自环L2的Phe203是精确的交联点。因此，我们的数据证实并扩展了基于RecA分子结构对RecA蛋白DNA结合结构域的预测（斯托里，R.M.，韦伯，I.T.，和斯蒂茨，T.A.（1992年）《自然》355，318 - 325）。
