The Integration Host Factor (IHF) Affects the Expression of the Phosphate-Binding Protein and of Alkaline Phosphatase in Escherichia coli

The genes encoding alkaline phosphatase (phoA) and the inducible inorganic phosphate transport system Pst (pstS,C,A,B,U) belong to the PHO regulon. Mutants of Escherichia coli lacking the global regulatory protein integration host factor (IHF) show an increased level of alkaline phosphatase and a decreased level of Pst. IHF binds weakly but specifically to a DNA fragment containing the promoter region of the pst operon but does not bind to a fragment that includes the promoter region of phoA. It is proposed that IHF is a positive regulator of the pst operon and as such controls indirectly the expression of phoA. Received: 4 May 1998 / Accepted: 19 August 1998     

Integration Host Factor of Mycobacterium tuberculosis,mIHF, Compacts DNA by a Bending Mechanism

The bacterial chromosomal DNA is folded into a compact structure called as ‘nucleoid’ so that the bacterial genome can be accommodated inside the cell. The shape and size of the nucleoid are determined by several factors including DNA supercoiling, macromolecular crowding and nucleoid associated proteins (NAPs). NAPs bind to different sites of the genome in sequence specific or non-sequence specific manner and play an important role in DNA compaction as well as regulation. Until recently, few NAPs have been discovered in mycobacteria owing to poor sequence similarities with other histone-like proteins of eubacteria. Several putative NAPs have now been identified in Mycobacteria on the basis of enriched basic residues or histone-like “PAKK” motifs. Here, we investigate mycobacterial Integration Host Factor (mIHF) for its architectural roles as a NAP using atomic force microscopy and DNA compaction experiments. We demonstrate that mIHF binds DNA in a non-sequence specific manner and compacts it by a DNA bending mechanism. AFM experiments also indicate a dual architectural role for mIHF in DNA compaction as well as relaxation. These results suggest a convergent evolution in the mechanism of E. coli and mycobacterial IHF in DNA compaction.     

Purification of Host DNA Synthesis-Suppressing Factor (DSF) Produced by Infection with Measles Virus

Host DNA synthesis-suppressing factor (DSF) produced into culture fluid of cloned HeLa cells (HeLa C-9) infected with a small plaque variant of Toyoshima strain of measles virus was purified by precipitation with ammonium sulfate, chromatography on CM-cellulose and DEAE-cellulose, and gel-filtration on Sephadex G-100 and G-200. The specific activity of the finally purified DSF was 302 units/mg of protein representing approximately 300-fold purification. The molecular weight of DSF was estimated to be about 55 000. By isoelectric focusing, two kinds of DSF having isoelectric points of 4.24 and 5.24 were detectable. The purified DSF was able to suppress host DNA synthesis of HeLa cells, continuous human lymphoid cells (NC-37), mouse L cells and Meth-A cells derived from an ascitic tumor of the mouse. The activity of the purified DSF was inactivated by heating at 56 C for 30 min or by treatment with trypsin.     

Integration host factor (IHF) stimulates binding of the gpNu1 subunit of lambda terminase to cos DNA.

The lambda terminase enzyme binds to the cohesive end sites (cos) of multimeric replicating lambda DNA and introduces staggered nicks to regenerate the 12 bp single-stranded cohesive ends of the mature phage genome. In vitro this endonucleolytic cleavage requires spermidine, magnesium ions, ATP and a host factor. One of the E. coli proteins which can fulfill this latter requirement is Integration Host Factor (IHF). IHF and the gpNu1 subunit of terminase can bind simultaneously to their own specific binding sites at cos. DNase I footprinting experiments suggest that IHF may promote gpNu1 binding. Although no specific gpNu1 binding to the left side of cos can be detected, this DNA segment does play a specific role since a cos fragment that does not include the left side or whose left side is replaced by non-cos sequences, is unable to bind gpNu1 unless either spermidine or IHF is present. Binding studies on the right side of cos using individual or combinations of gpNu1 binding sites I, II and III indicate that binding at sites I and II is not optimal unless site III is present.     

In Vitro Selection of Integration Host Factor Binding Sites

Integration host factor (IHF) is a bacterial protein that binds and severely bends a specific DNA target. IHF binding sites are approximately 30 to 35 bp long and are apparently divided into two domains. While the 3' domain is conserved, the 5' domain is degenerate but is typically AT rich. As a result of physical constraints that IHF must impose on DNA in order to bind, it is believed that this 5' domain must possess structural characteristics conducive for both binding and bending with little regard for specific contacts between the protein and the DNA. We have examined the sequence requirements of the 5' binding domain of the IHF binding target. Using a SELEX procedure, we randomized and selected variants of a natural IHF site. We then analyzed these variants to determine how the 5' binding domain affects the structure, affinity, and function of an IHF-DNA complex in a native system. Despite finding individual sequences that varied over 100-fold in affinity for IHF, we found no apparent correlation between affinity and function.     

Site-Specific Recombination of Bacteriophage P22 Does Not Require Integration Host Factor

Site-specific recombination by phages lambda and P22 is carried out by multiprotein-DNA complexes. Integration host factor (IHF) facilitates lambda site-specific recombination by inducing DNA bends necessary to form an active recombinogenic complex. Mutants lacking IHF are over 1,000-fold less proficient in supporting lambda site-specific recombination than wild-type cells. Although the attP region of P22 contains strong IHF binding sites, in vivo measurements of integration and excision frequencies showed that infecting P22 phages can perform site-specific recombination to its maximum efficiency in the absence of IHF. In addition, a plasmid integration assay showed that integrative recombination occurs equally well in wild-type and ihfA mutant cells. P22 integrative recombination is also efficient in Escherichia coli in the absence of functional IHF. These results suggest that nucleoprotein structures proficient for recombination can form in the absence of IHF or that another factor(s) can substitute for IHF in the formation of complexes.     

Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction

The annotated whole-genome sequence of Mycobacterium tuberculosis revealed that Rv1388 (Mtihf) is likely to encode for a putative 20-kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or the organization of the mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg-170, Arg-171, and Arg-173, which might be involved in DNA binding, and a conserved proline (Pro-150) in the tight turn. The phenotypic sensitivity of Escherichia coli ΔihfA and ΔihfB strains to UV and methyl methanesulfonate could be complemented with the wild-type Mtihf but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, binds with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHFαβ. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. We therefore propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.     

HAP转录因子与DNA结合的活力受细胞内氧化还原系统的调节

ＨＡＰ 转录因子（ ＨＡＰ２／ＨＡＰ３／ＨＡＰ４／ＨＡＰ５） 是存在于酿酒酵母中的一种异源多聚蛋白,它能与酵母中许多启动子上游的ＣＣＡＡＴ盒（ 顺式作用元件） 专一性结合, 以增强基因的转录。在酵母ｈａｐ５ 突变株的细胞中,用酵母单杂交系统从水稻ｃＤＮＡＧＡＬ４ 表达文库中筛选出的阳性克隆是编码谷胱甘肽氧还蛋白的ｃＤＮＡ,提示细胞内的氧化还原系统可能作用于ＨＡＰ蛋白,从而对ＣＣＡＡＴ盒的结合活力起调节作用。对ＨＡＰ３ 亚基分子中半胱氨酸残基的突变实验结果支持上述推测     

Patterns of Viral DNA Integration in Cells Transformed by Wild Type or DNA-Binding Protein Mutants of Adenovirus Type 5 and Effect of Chemical Carcinogens on Integration

The integration pattern of viral DNA was studied in a number of cell lines transformed by wild-type adenovirus type 5 (Ad5 WT) and two mutants of the DNA-binding protein gene, H5ts125 and H5ts107. The effect of chemical carcinogens on the integration of viral DNA was also investigated. Liquid hybridization (C(0)t) analyses showed that rat embryo cells transformed by Ad5 WT usually contained only the left-hand end of the viral genome, whereas cell lines transformed by H5ts125 or H5ts107 at either the semipermissive (36 degrees C) or nonpermissive (39.5 degrees C) temperature often contained one to five copies of all or most of the entire adenovirus genome. The arrangement of the integrated adenovirus DNA sequences was determined by cleavage of transformed cell DNA with restriction endonucleases XbaI, EcoRI, or HindIII followed by transfer of separated fragments to nitrocellulose paper and hybridization according to the technique of E. M. Southern (J. Mol. Biol. 98: 503-517, 1975). It was found that the adenovirus genome is integrated as a linear sequence covalently linked to host cell DNA; that the viral DNA is integrated into different host DNA sequences in each cell line studied; that in cell lines that contain multiple copies of the Ad5 genome the viral DNA sequences can be integrated in a single set of host cell DNA sequences and not as concatemers; and that chemical carcinogens do not alter the extent or pattern of viral DNA integration.     

Structural Mechanisms of Cooperative DNA Binding by Bacterial Single-Stranded DNA-Binding Proteins

Bacteria encode homooligomeric single-stranded (ss) DNA-binding proteins (SSBs) that coat and protect ssDNA intermediates formed during genome maintenance reactions. The prototypical Escherichia coli SSB tetramer can bind ssDNA using multiple modes that differ by the number of bases bound per tetramer and the magnitude of the binding cooperativity. Our understanding of the mechanisms underlying cooperative ssDNA binding by SSBs has been hampered by the limited amount of structural information available for interfaces that link adjacent SSB proteins on ssDNA. Here we present a crystal structure of Bacillus subtilis SsbA bound to ssDNA. The structure resolves SsbA tetramers joined together by a ssDNA “bridge” and identifies an interface, termed the “bridge interface,” that links adjacent SSB tetramers through an evolutionarily conserved surface near the ssDNA-binding site. E. coli SSB variants with altered bridge interface residues bind ssDNA with reduced cooperativity and with an altered distribution of DNA binding modes. These variants are also more readily displaced from ssDNA by RecA than wild-type SSB. In spite of these biochemical differences, each variant is able to complement deletion of the ssb gene in E. coli. Together our data suggest a model in which the bridge interface contributes to cooperative ssDNA binding and SSB function but that destabilization of the bridge interface is tolerated in cells.     

The Pseudomonas aeruginosa hemA promoter is regulated by Anr,Dnr, NarL and Integration Host Factor

Pseudomonas aeruginosa forms most of its heme under anaerobic denitrifying conditions. To study the regulation of the hemA gene, which codes for the first enzyme of heme biosynthesis in P. aeruginosa, a lacZ reporter gene fusion was constructed. Expression of lacZ under the control of the hemA promoter was found to be increased by 2.8-fold under anaerobic conditions in the presence of the alternative electron acceptor nitrate, relative to the level observed under aerobic growth conditions. Anaerobic fermentative growth or the presence of nitrite did not affect the lacZ expression. The genes encoding the oxygen sensor protein Anr, the redox regulator Dnr, the nitrate regulator NarL and the DNA-bending Integration Host Factor (IHF) are all required for the cooperative anaerobic induction of the hemA promoter hemAp (1). Potential binding sites for these regulatory proteins were identified by site-directed mutagenesis of the promoter fused to the reporter gene. The mode of regulation of P. aeruginosa hemA differs significantly from that described for the hemA gene of Escherichia coli K-12.     

Colocalization of Multiple DNA Loci: A Physical Mechanism

A variety of important cellular processes require, for functional purposes, the colocalization of multiple DNA loci at specific time points. In most cases, the physical mechanisms responsible for bringing them in close proximity are still elusive. Here we show that the interaction of DNA loci with a concentration of diffusing molecular factors can induce spontaneously their colocalization, through a mechanism based on a thermodynamic phase transition. We consider up to four DNA loci and different valencies for diffusing molecular factors. In particular, our analysis illustrates that a variety of nontrivial stable spatial configurations is allowed in the system, depending on the details of the molecular factor/DNA binding-sites interaction. Finally, we discuss as a case study an application of our model to the pairing of X chromosome at X inactivation, one of the best-known examples of DNA colocalization. We also speculate on the possible links between X colocalization and inactivation.