Replication Protein A Interactions with DNA. 2. Characterization of Double-Stranded DNA-Binding/Helix-Destabilization Activities and the Role of the Zinc-Finger Domain in DNA Interactions
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- 作者：Ye Lao ; Chang Geun Lee ; and Marc S. Wold
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：March 30, 1999
- 年：1999
- 卷：38
- 期：13
- 页码：3974 - 3984
- 全文大小：183K
- 年卷期：v.38,no.13(March 30, 1999)
- ISSN：1520-4995
文摘
Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding proteinthat is composed of subunits of 70, 32, and 14 kDa. RPA is required for multiple processes in cellularDNA metabolism. RPA has been reported to (1) bind with high affinity to single-stranded DNA (ssDNA),(2) bind specifically to certain double-stranded DNA (dsDNA) sequences, and (3) have DNA helix-destabilizing ("unwinding") activity. We have characterized both dsDNA binding and helix destabilization.The affinity of RPA for dsDNA was lower than that of ssDNA and precisely correlated with the meltingtemperature of the DNA fragment. The rates of helix destabilization and dsDNA binding were similar,and both were slow relative to the rate of binding ssDNA. We have previously mapped the regions requiredfor ssDNA binding [Walther et al. (1999) Biochemistry 38, 3963-3973]. Here, we show that both helix-destabilization and dsDNA-binding activities map to the central DNA-binding domain of the 70-kDasubunit and that other domains of RPA are needed for optimal activity. We conclude that all types ofRPA binding are manifestations of RPA ssDNA-binding activity and that dsDNA binding occurs whenRPA destabilizes a region of dsDNA and binds to the resulting ssDNA. The 70-kDa subunit of all RPAhomologues contains a highly conserved putative (C-X2-C-X13-C-X2-C) zinc finger. This motif directlyinteracts with DNA and contributes to dsDNA-binding/unwinding activity. Evidence is presented that ametal ion is required for the function of the zinc-finger motif.