A C-terminal segment with properties of alpha-helix is essential for DNA binding and in vivo function of zinc finger protein Rme1p

Rme1p plays important roles in the control of meiosis and in cell cycle progression through binding to upstream regions of IME1 and CLN2 in Saccharomyces cerevisiae. Rme1p has three zinc finger segments, and two of them are atypical. To determine DNA binding domain of Rme1p, a series of Rme1p derivatives fused with maltose-binding protein were purified and characterized by gel mobility shift assay. We show that not only three zinc fingers, but also the neighboring C-terminal region is essential for DNA binding. Mutational analysis of this region revealed that basic residues Arg-287, Lys-290, and Arg-291 and the hydrophobic residues Phe-288, Leu-292, Ile-295, and Leu-296 are critical for DNA binding. In addition, double substitutions by proline at Asn-289 and Lys-293, each of which was not essential for DNA binding, abolished DNA binding. These results suggest that the C-terminal segment forms an amphipathic helical structure. Furthermore, it was shown that the mutations in the important basic residues abolish or impair Rme1p function in vivo for repression and inhibition of spore formation. Thus, the C-terminal segment is essential and acts as a novel accessory domain for DNA binding by zinc fingers.     

Genomic footprinting of the yeast zinc finger protein Rme1p and its roles in repression of the meiotic activator IME1.

The zinc finger protein Rme1p is a negative regulator of the meiotic activator IME1 in Saccharomyces cerevisiae . Prior studies have shown that Rme1p binds in vitro to a site near nt -2030 in the IME1 upstream region, but a genomic mutation in that site has little effect on repression of IME1 . To identify Rme1p binding sites in vivo , we have examined the binding of Rme1p to genomic sites through in vivo footprinting. We show that Rme1p binds to two sites in the IME1 upstream region, near nt -1950 and -2030. Mutations in both binding sites abolish repression of chromosomal IME1 by Rme1p, whereas a mutation in either single site causes partial derepression. Therefore, both Rme1p binding sites are essential for repression of IME1 . Prior studies have shown that repression by Rme1p depends upon RGR1 and SIN4 , which specify RNA polymerase II mediator subunits that are required for normal nucleosome density. We find that RGR1 and SIN4 are not simply required for Rme1p to bind to DNA in vivo . These results suggest that Rme1p functions directly as a repressor of IME1 and that Rgr1p and Sin4p are required for DNA-bound Rme1p to exert repression.     

大肠杆菌YacG锌指结构的金属结合及功能特性

YacG蛋白是一种能够抑制大肠杆菌促旋酶（E.coli gyrase）活性的内源性小分子蛋白质,仅由65 个氨基酸残基组成。核磁共振(NMR)研究发现,YacG结构中含有1个Cys-X2-Cys-X15-Cys-X3-Cys序列的锌指结构域,然而其作用并不清楚。本研究发现,在添加外源锌或者铁的M9基础培养基中,表达并纯化得到分别含有锌和铁的YacG蛋白,而在同时添加铁和L-半胱氨酸的M9基础培养基中可以纯化得到含有铁硫簇的蛋白质。这表明,YacG不仅是一个锌指蛋白,也是铁结合或铁硫簇结合蛋白。定点突变实验发现,YacG锌指结构中的4个半胱氨酸残基突变后,其结合的锌、铁、铁硫簇的含量都显著下降。这提示,锌结合、铁结合以及铁硫簇结合的位点均位于锌指结构域中的4个半胱氨酸残基。体内YacG过表达实验显示,用IPTG在大肠杆菌体内诱导表达野生型YacG蛋白会导致其生长明显受到抑制,而过表达突变体蛋白（YacG-C12/28S）对其生长的抑制作用将会减弱。体外实验进一步发现,锌结合、铁结合以及铁硫簇结合形式的YacG蛋白对E.coli gyrase促DNA螺旋活性的抑制作用没有明显差别,但是锌指结构突变体蛋白（YacG-C12/28S）对gyrase活性的抑制作用显著减弱。这说明,完整的锌指结构对YacG抑制gyrase活性的功能具有重要作用。此研究有可能为gyrase抑制剂类抗生素药物的研发提供有用的线索。     

Involvement of lysine residues 289 and 291 of the cAMP-responsive element-binding protein in the recognition of the cAMP-responsive element.

The molecular interactions resulting in specific binding of trans-acting factors to distinct cis-acting elements is not well understood. Here we report our attempt to understand the involvement of distinct amino acid residues of the basic domain of cAMP-responsive element-binding protein (delta CREB) in the determination of binding toward the cAMP-responsive element (CRE). Using in vitro mutagenesis, we constructed site-directed mutants of distinct amino acid residues within the DNA contact region of delta CREB. The activities of the mutant proteins were analyzed by gel retardation, methylation interference, and CRE competition analyses. We demonstrate that a single lysine to glutamine substitution at positions 289 and 291 of delta CREB alters the methylation interference pattern of the mutant protein for the CRE site. Additional mutants constructed at these positions demonstrate that only identical basic residues at both positions 289 and 291 of delta CREB can restore the wild type methylation interference pattern of the mutant delta CREB protein for the CRE site. These observations point to the importance of the lysine residues at positions 289 and 291 in the process of CRE binding. In addition, this observation suggests that the symmetrical nature of the CRE site is reflected in the DNA contact region of the protein.     

Characterization of the minimal DNA-binding domain of the HIV integrase protein.

The human immunodeficiency virus (HIV) integrase (IN) protein mediates an essential step in the retroviral lifecycle, the integration of viral DNA into human DNA. A DNA-binding domain of HIV IN has previously been identified in the C-terminal part of the protein. We tested truncated proteins of the C-terminal region of HIV-1 IN for DNA binding activity in two different assays: UV-crosslinking and southwestern blot analysis. We found that a polypeptide fragment of 50 amino acids (IN220-270) is sufficient for DNA binding. In contrast to full-length IN protein, this domain is soluble under low salt conditions. DNA binding of IN220-270 to both viral DNA and non-specific DNA occurs in an ion-independent fashion. Point mutations were introduced in 10 different amino acid residues of the DNA-binding domain of HIV-2 IN. Mutation of basic amino acid K264 results in strong reduction of DNA binding and of integrase activity.     

Mechanism of DNA binding by the DnaB helicase of Escherichia coli: analysis of the roles of domain gamma in DNA binding.

We have analyzed the mechanism of single-stranded DNA (ssDNA) binding mediated by the C-terminal domain gamma of the DnaB helicase of Escherichia coli. Sequence analysis of this domain indicated a specific basic region, "RSRARR", and a leucine zipper motif that are likely involved in ssDNA binding. We have carried out deletion as well as in vitro mutagenesis of specific amino acid residues in this region in order to determine their function(s) in DNA binding. The functions of the RSRARR domain in DNA binding were analyzed by site-directed mutagenesis. DnaBMut1, with mutations R(328)A and R(329)A, had a significant decrease in the DNA dependence of ATPase activity and lost its DNA helicase activity completely, indicating the important roles of these residues in DNA binding and helicase activities. DnaBMut2, with mutations R(324)A and R(326)A, had significantly attenuated DNA binding as well as DNA-dependent ATPase and DNA helicase activities, indicating that these residues also play a role in DNA binding and helicase activities. The role(s) of the leucine zipper dimerization motif was (were) determined by deletion analysis. The DnaB Delta 1 mutant with a 55 amino acid C-terminal deletion, which left the leucine zipper and basic DNA binding regions intact, retained DNA binding as well as DNA helicase activities. However, the DnaB Delta 2 mutant with a 113 amino acid C-terminal deletion that included the leucine zipper dimerization motif, but not the RSRARR sequence, lost DNA binding, DNA helicase activities, and hexamer formation. The major findings of this study are (i) the leucine zipper dimerization domain, I(361)-L(389), is absolutely required for (a) dimerization and (b) ssDNA binding; (ii) the base-rich RSRARR sequence is required for DNA binding; (iii) three regions of domain gamma (gamma I, gamma II, and gamma III) differentially regulate the ATPase activity; (iv) there are likely three ssDNA binding sites per hexamer; and (v) a working model of DNA unwinding by the DnaB hexamer is proposed.     

Analysis of wild-type and mutant p21WAF-1 gene activities.

The p21WAF-1 gene is positively regulated by the wild-type p53 protein. p21WAF-1 has been shown to interact with several cyclin-dependent kinase complexes and block the activity of G1 cyclin-dependent kinases (cdks). Mutational analysis with the p21WAF-1 gene localized a site, at amino acid residues 21 and 24 in the amino terminus of the protein, for p21WAF-1 binding to cyclins D and E. This region of the protein is conserved (residues 21 to 26) in other p21WAF-1 family members, p27kip-1 and p57kip-2. The same p21WAF-121,24 mutant also fails to bind to cyclin D1-cdk 4 or cyclin E-cdk 2 complexes in vitro, suggesting that amino acid residues 21 and 24 are important for p21WAF-1-cdk-cyclin trimeric complex interactions. The p21WAF-1 wild-type protein will suppress tumor cell growth in culture while p21WAF-1 mutant proteins with defects in residues 21 and 24 fail to suppress tumor cell growth. The overexpression of cyclin D or E in these cells will partially overcome the growth suppression of wild-type p21WAF-1 protein in cells. These results provide evidence that p21WAF-1 acts through cyclin D1-cdk4 and cyclin E-cdk2 complexes in vivo to induce the growth suppression. The p21WAF-1 binding sites for cyclins (residues 21 to 26), cdk2 (residues 49 to 71), and proliferating-cell nuclear antigen (residues 124 to 164) have all been mapped to discrete sites on the protein.     

The loop region of the helix-loop-helix protein Id1 is critical for its dominant negative activity.

Id1, a helix-loop-helix (HLH) protein which lacks a DNA binding domain, has been shown to negatively regulate other members of the HLH family by direct protein-protein interactions, both in vitro and in vivo. In this study, we report the results of site-directed mutagenesis experiments aimed at defining the regions of Id1 which are important for its activity. We have found that the HLH domain of Id1 is necessary and nearly sufficient for its activity. In addition, we show that two amino acid residues at the amino terminus of the Id1 loop are critical for its activity, perhaps by specifying the correct dimerization partners. In this regard, replacing the first four amino acids of the loops of the basic HLH proteins E12 and E47 with the corresponding amino acids of Id1 confers Id1 dimerization specificity. These studies point to the loop region as an important structural and functional element of the Id subfamily of HLH proteins.     

Analysis of functional domains of Rts1 RepA by means of a series of hybrid proteins with P1 RepA.

The RepA protein of the plasmid Rts1, consisting of 288 amino acids, is a trans-acting protein essential for initiation of plasmid replication. To study the functional domains of RepA, hybrid proteins of Rts1 RepA with the RepA initiator protein of plasmid P1 were constructed such that the N-terminal portion was from Rts1 RepA and the C-terminal portion was from P1 RepA. Six hybrid proteins were examined for function. The N-terminal region of Rts1 RepA between amino acid residues 113 and 129 was found to be important for Rts1 ori binding in vitro. For activation of the origin in vivo, an Rts1 RepA subregion between residues 177 and 206 as well as the DNA binding domain was required. None of the hybrid initiator proteins activated the P1 origin. Both in vivo and in vitro studies showed, in addition, that a C-terminal portion of Rts1 RepA was required along with the DNA binding and ori activating domains to achieve autorepression, suggesting that the C-terminal region of Rts1 RepA is involved in dimer formation. A hybrid protein consisting of the N-terminal 145 amino acids of Rts1 and the C-terminal 142 amino acids from P1 showed strong interference with both Rts1 and P1 replication, whereas other hybrid proteins showed no or little effect on P1 replication.     

Single exchanges of amino acids in the basic region change the specificity of N-Myc.

We exchanged specific amino acids in the basic region of the murine N-Myc protein and tested the mutant proteins for their DNA binding specificity. The amino acids we exchanged were chosen in analogy to residues of the homologous basic regions of bHLH and bZIP proteins. Mutant N-Myc peptides were expressed in Escherichia coli and specific DNA binding was monitored by gel shift experiments. For this we used palindromic target sequences with systematic base pair exchanges. Several mutants with altered DNA binding specificity were identified. Amino acid exchanges of residues -14 or -10 of the basic region lead to specificity changes (we define leucine 402 of N-Myc as +1; comparable to GCN4 see (1)). The palindromic N-Myc recognition sequence 5'CACGTG is no longer recognized by the mutant proteins, but DNA fragments with symmetrical exchanges of the target sequence are. Exchanges at position -15 broaden the binding specificity. These data were used to build a computer based model of the putative interactions of the N-Myc basic DNA binding region with its target sequence.     

Role of the N-terminal domain of the human DMC1 protein in octamer formation and DNA binding

The DMC1 protein, a eukaryotic homologue of RecA that shares significant amino acid identity with RAD51, exhibits two oligomeric DNA binding forms, an octameric ring and a helical filament. In the crystal structure of the octameric ring form, the DMC1 N-terminal domain (1-81 amino acid residues) was highly flexible, with multiple conformations. On the other hand, the N-terminal domain of Rad51 makes specific interactions with the neighboring ATPase domain in the helical filament structure. To gain insights into the functional role of the N-terminal domain of DMC1, we prepared a deletion mutant, DMC1-(82-340), that lacks the N-terminal 81 amino acid residues from the human DMC1 protein. Analytical ultracentrifugation experiments revealed that, whereas full-length DMC1 forms a octamer, DMC1-(82-340) is a heptamer. Furthermore, DNA binding experiments showed that DMC1-(82-340) was completely defective in both single-stranded and double-stranded DNA binding activities. Therefore, the N-terminal domain of DMC1 is required for the formation of the octamer, which may support the proper DNA binding activity of the DMC1 protein.     

Structure-Based Mutational Analysis of the C-Terminal DNA-Binding Domain of Human Immunodeficiency Virus Type 1 Integrase: Critical Residues for Protein Oligomerization and DNA Binding

The C-terminal domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) is a dimer that binds to DNA in a nonspecific manner. The structure of the minimal region required for DNA binding (IN_220–270) has been solved by nuclear magnetic resonance spectroscopy. The overall fold of the C-terminal domain of HIV-1 IN is similar to those of Src homology region 3 domains. Based on the structure of IN_220–270, we studied the role of 15 amino acid residues potentially involved in DNA binding and oligomerization by mutational analysis. We found that two amino acid residues, arginine 262 and leucine 234, contribute to DNA binding in the context of IN_220–270, as indicated by protein-DNA UV cross-link analysis. We also analyzed mutant proteins representing portions of the full-length IN protein. Amino acid substitution of residues located in the hydrophobic dimer interface, such as L241A and L242A, results in the loss of oligomerization of IN; consequently, the levels of 3′ processing, DNA strand transfer, and intramolecular disintegration are strongly reduced. These results suggest that dimerization of the C-terminal domain of IN is important for correct multimerization of IN.