Type I restriction enzymes bind to a specific DNA sequence and subsequently translocate DNA past the complex to reach a non-specific cleavage site. We have examined several potential blocks to DNA translocation, such as positive supercoiling or a Holliday junction, for their ability to trigger DNA cleavage by type I restriction enzymes. Introduction of positive supercoiling into plasmid DNA did not have a significant effect on the rate of DNA cleavage by EcoAI endonuclease nor on the enzyme's ability to select cleavage sites randomly throughout the DNA molecule. Thus, positive supercoiling does not prevent DNA translocation. EcoR124II endonuclease cleaved DNA at Holliday junctions present on both linear and negatively supercoiled substrates. The latter substrate was cleaved by a single enzyme molecule at two sites, one on either side of the junction, consistent with a bi-directional translocation model. Linear DNA molecules with two recognition sites for endonucleases from different type I families were cut between the sites when both enzymes were added simultaneously but not when a single enzyme was added. We propose that type I restriction enzymes can track along a DNA substrate irrespective of its topology and cleave DNA at any barrier that is able to halt the translocation process.

We have characterized a novel mutant of EcoDXXI, a type IC DNA restriction and modification (R‐M) system, in which the specificity has been altered due to a Tn5 insertion into the middle of hsdS, the gene which encodes the polypeptide that confers DNA sequence specificity to both the restriction and the modification reactions. Like other type I enzymes, the wild type EcoDXXI recognizes a sequence composed of two asymmetrical half sites separated by a spacer region: TCA(N7)RTTC. Purification of the EcoDXXI mutant methylase and subsequent in vitro DNA methylation assays identified the mutant recognition sequence as an interrupted palindrome, TCA(N8)TGA, in which the 5′ half site of the wild type site is repeated in inverse orientation. The additional base pair in the non‐specific spacer of the mutant recognition sequence maintains the proper spacing between the two methylatable adenine groups. Sequencing of both the wild type and mutant EcoDXXI hsdS genes showed that the Tn5 insertion occurred at nucleotide 673 of the 1221 bp gene. This effectively deletes the entire carboxyl‐terminal DNA binding domain which recognizes the 3′ half of the EcoDXXI binding site. The truncated hsdS gene still encodes both the amino‐terminal DNA binding domain and the conserved repeated sequence that defines the length of the recognition site spacer region. We propose that the EcoDXXI mutant methylase utilizes two truncated hsdS subunits to recognize its binding site. The implications of this finding in terms of subunit interactions and the malleability of the type I R‐M systems will be discussed.

The bacteriophage P22-based challenge phage system was used to study the binding of integration host factor (IHF) to its H' recognition site in the attP region of bacteriophage lambda. We constructed challenge phages that carried H' inserts in both orientations within the P22 Pa", promoter, which is required for antirepressor synthesis. We found that IHF repressed expression of Pa", from either challenge phage when expressed from an inducible P,,, promoter on a plasmid vector. Mutants containing changes in the H' inserts that decrease or eliminate IHF binding were isolated by selecting challenge phages that could synthesize antirepressor in the presence of IHF. Sequence analysis of 31 mutants showed that most changes were base pair substitutions within the H' insert. Approximately one-half of the mutants contained substitutions that changed base pairs that are part of the IMF consensus binding site; mutants were isolated that contained substitutions at six of the nine base pairs of the consensus site. Other mutants contained changes at base pairs between the two subdeterminants of the H' site, at positions that are not specffied in the consensus sequence, and in the dA+dT-rich region that flanks the consensus region of the site. Taken together, these results show that single-base-pair changes at positions outside of the proposed consensus bases can weaken or drastically disrupt I1F binding to the mutated site.The integration host factor (IHF) of Escherichia coli was originally discovered as a protein present in crude extracts that is required for integration of phage lambda DNA into the host chromosome (14, 15). More recently, it has been shown that IHF participates in a variety of other processes, including control of gene expression, plasmid replication, transposition, and the packaging of viral DNA into capsids (for a review, see reference 6).IHF is a sequence-specific DNA-binding protein, and a comparison of several IHF binding sites has led to the determination of a consensus recognition sequence; WATC AANNNNTTR (where W is A or T, N is any nucleotide, and R is A or G). In addition, IHF bends DNA upon binding (11,16,18,26,28,31,32); it is possible that its primary role in phage lambda recombination and other processes is to bend the DNA into a functional conformation (8,30).IHF is a heterodimer (Mr 20,000) composed of the gene products of the hip (himD) (Mr 9,000) and the himA (Mr 11,000) genes. Sequence analysis of the hip and himA (5, 21) genes showed that they are related to each other as well as to the HU protein and to transcription factor 1 of bacteriophage SPOl (for reviews, see references 4 and 10). The latter two proteins are basic and bind nonspecifically to DNA. The details of IHF binding to DNA remain to be elucidated, but recent footprinting studies indicate that IHF, unlike most sequence-specific binding proteins, interacts primarily with the minor groove of the DNA (3, 18, 33).Among the IHF binding sites, the three in the attP region of phage lambda (Hi, H2, and H') are the best characterized...

With the advent of the recent determination of high-resolution crystal structures of bovine rhodopsin and human beta2 adrenergic receptor (beta2AR), there are still many structure-function relationships to be learned from other G protein-coupled receptors (GPCRs). Many of the pharmaceutically interesting GPCRs cannot be modeled because of their amino acid sequence divergence from bovine rhodopsin and beta2AR. Structure determination of GPCRs can provide new avenues for engineering drugs with greater potency and higher specificity. Several obstacles need to be overcome before membrane protein structural biology becomes routine: over-expression, solubilization, and purification of milligram quantities of active and stable GPCRs. Coordinated iterative efforts are required to generate any significant GPCR over-expression. To formulate guidelines for GPCR purification efforts, we review published conditions for solubilization and purification using detergents and additives. A discussion of sample preparation of GPCRs in detergent phase, bicelles, nanodiscs, or low-density lipoproteins is presented in the context of potential structural biology applications. In addition, a review of the solubilization and purification of successfully crystallized bovine rhodopsin and beta2AR highlights tools that can be used for other GPCRs.

The hsdS subunit of a type IC restriction‐modification enzyme is responsible for the enzyme's DNA binding specificity. Type I recognition sites are characterized by two defined half‐sites separated by a non‐specific spacer of defined length. The hsdS subunit contains two independent DNA binding domains, each targeted towards one DNA half‐site. We have shown previously that the 5′ half of hsdS can code for a functional substitute of the full‐length hsdS. Here we demonstrate that the 3′ half of the gene, when fused to the appropriate transcriptional and translational start signals, also codes for a peptide which imparts DNA binding specificity to the enzyme. About half the natural hsdS size, the mutant peptide contains a single DNA recognition domain flanked by one copy of each internal repeat found in the full‐length hsdS. Deletion of either repeat sequence results in loss of activity. Like the 5′ hsdS mutant, the 3′ mutant recognizes an interrupted palindrome, GAAYN(5)RTTC, suggesting that two truncated subunits participate in DNA recognition. Co‐expression of the 5′ hsdS mutant and the 3′ hsdS mutant along with hsdM regenerates the wild‐type methylation specificity. Thus, there is a free assortment of subunits in the cell.