Primary structure of the hip gene of Escherichia coli and of its product, the beta subunit of integration host factor.

We describe the isolation and sequencing of the hip gene of Escherichia coli and show that it encodes the beta subunit of integration host factor (IHF beta). In order to locate the coding region, we constructed a set of deletion mutants by exonucleolytic digestion of a fragment containing hip, determined which mutants were hip+ and which hip- by complementation, and then sequenced the ends of the critical deletions. The 5' end of the coding region was located precisely by comparing the deduced amino acid sequence to the actual N-terminal amino acid sequence of IHF. Our assignment of the coding region was further substantiated by the nucleotide sequences of a hip point mutant and of internal replacement mutations. We found a probable promoter for hip located about 85 base-pairs upstream from the initial AUG codon and about 75 base-pairs downstream from the 3' end of the neighboring gene, rpsA, and we constructed an IHF beta overproducer by fusing the coding sequences to the lambda pL promoter. A survey of known protein sequences revealed a close relationship between IHF beta and the type II prokaryotic DNA binding proteins (the "histone-like" proteins). This relationship is shared to a considerable extent by the other subunit of IHF, IHF alpha. A hip missense mutation that replaces a completely conserved glycine with aspartate has a null phenotype, suggesting that the conserved regions are functionally important.

Identifying determinants of recombination specificity: construction and characterization of chimeric bacteriophage integrases.

Bacteriophage integrases are members of a family of structurally related enzymes that promote recombination between DNA molecules that carry specific sites. Phages lambda and HK022 encode closely related integrases that recognize different sets of sequences within the core regions of their respective attachment sites. To locate the amino acid residues that determine this difference in specificity, we isolated recombinant phages that produce chimeric integrases and measured the ability of these chimeras to promote recombination of lambda and HK022 sites in vivo. A chimera that is of lambda origin except for one HK022 residue at position 99 and 12 HK022 residues located between positions 279 and 329 had wild-type HK022 specificity and activity for both integrative and excisive recombination. Chimeras containing certain subsets of these 13 residues had incomplete specificity. The region around position 99 is not well-conserved in other members of the integrase family, but the 279-329 segment includes residues that are highly conserved and believed to be directly involved in catalysis. Many chimeras were inactive in recombining either HK022 or lambda sites. Selection for mutants that restored activity to these chimeras revealed sets of residues that are likely to interact with each other.

Identifying determinants of recombination specificity: construction and characterization of mutant bacteriophage integrases.

The Integrases of bacteriophages lambda and HK022 promote recombination between DNA molecules that carry attachment sites. The two integrases are about 70% identical in sequence and catalyze nearly identical reactions, but recognize different sets of sites. To identify the amino acids that determine this difference in specificity, we selected mutants of lambda integrase with increased ability to recombine HK022 sites. This selection yielded eleven different amino acid substitutions at eight different positions. Three of the positions belong to a larger set that were identified as important for the lambda/HK022 specificity difference by analysis of chimeric integrases. Substitution of the HK022 for the corresponding lambda residue at each of these three positions increased recombination of HK022 sites, and one double substitution, N99D-E319R, increased recombination to nearly wild-type HK022 levels. Mutations at the other five positions changed residues that are identical in the wild-type proteins or are at positions identified by chimera analysis as unimportant for the lambda/HK022 specificity difference. All of the mutants isolated by selection for increased recombination of HK022 sites retained considerable ability to recombine lambda sites. However, we found that substitution of HK022 for lambda residues at three additional positions, S282P, G283K, and R287K, specifically reduced recombination of lambda sites. These three substitutions when combined with N99D and E319R were sufficient to change the specificity of lambda to that of HK022 integrase. The first three substitutions act principally to prevent recombination of lambda sites, and the second two to remove a barrier to recombination of HK022 sites. We suggest that many natural alterations in the specificity of protein-DNA interactions occur by multi-step changes that first relax and then restrict specificity.

A zinc-binding region in the beta' subunit of RNA polymerase is involved in antitermination of early transcription of phage HK022.

Antitermination of early transcription in phage HK022 requires no virus-encoded proteins and thus differs from antitermination by other lambdoid phages. It does require cis-acting phage sequences, which may be analogous to the lambdoid nut sites. To identify host proteins involved in antitermination, we isolated 14 Escherichia coli mutants that are specifically blocked in HK022 growth. The mutations are located in the rpoC gene, which encodes the beta' subunit of RNA polymerase. Each mutation alters one of three amino acid residues located within a cluster of four completely conserved cysteine residues that are believed to bind zinc. We examined the effect of one mutation on HK022 antitermination in vivo. rpoCY75N greatly reduced readthrough of a strong rho-independent transcription terminator placed downstream of the HK022 PL promoter and nutL analog, but did not decrease promoter activity. Purified enzyme had a similar effect on PL-directed transcription in vitro: wild-type but not mutant polymerase read through a strong rho-independent terminator located immediately downstream of the nutL analog with high efficiency. We suggest that interaction of the putative zinc-binding domain of the RNA polymerase beta' subunit with the HK022 antitermination sites suppresses transcription termination, and that this interaction can occur in the absence of other proteins.

Recognition of core binding sites by bacteriophage integrases.

Bacteriophage integrases promote recombination between DNA molecules that carry attachment sites. They are members of a large and widely distributed family of site-specific recombinases with diverse biological roles. The integrases of phages lambda and HK022 are closely related members of this family, but neither protein efficiently recombines the attachment sites of the other phage. The nucleotides responsible for this specificity difference are located close to the points of recombinational strand exchange, within an integrase binding motif called the extended core binding site. There are four imperfectly repeated copies of this motif in each set of phage attachment sites, but only two, B' and C, contain major specificity determinants. When these specificity determinants were replaced by the corresponding nucleotides from a site with the alternative specificity, the resulting mutant was recombined by both integrases. Thus, the determinants act by impeding recombination promoted by the non-cognate integrase. We found that identical nucleotide substitutions within different core site copies had different effects on recombination, suggesting that integrase does not recognize each of the extended core binding sites in the same way. Finally, substitution at several positions in lambda integrase with the corresponding HK022-specific amino acids prevents recombination of lambda attachment sites, and this defect can be suppressed in an allele-specific manner by appropriate substitutions of HK022-specific nucleotides in the extended core binding sites.

Lambda nutR mutations convert HK022 Nun protein from a transcription termination factor to a suppressor of termination.

The Nun protein of the lambdoid phage HK022 blocks lambda growth by terminating transcription at (or near) the lambda nut sites. An HK022 lysogen carrying a fusion of the lambda pR promoter and nutR site to a gal operon that lacks its own promoter is, therefore, Gal-. To characterize the target of Nun action, spontaneous Gal+ revertants of this strain were isolated and characterized. Two cis-acting mutations are located in the fusion and represent transversions of conserved nucleotides within the boxA sequence (CGCTCTTA) of nutR. One mutation, (CTCTCTTA), is identical with boxA5. The second, boxA16 (CGCTATTA), has not been reported previously. In the absence of Nun, both boxA mutants reduce gal expression. Analysis of in vivo fusion RNA indicates that the mutations increase termination at or near tR1, a rho-dependent lambda terminator located upstream from the fusion point. In contrast to the nutR+ fusion, Nun stimulates gal expression in the boxA mutants by suppressing transcription termination in the tR1 region. Nun antitermination, however, does not extend to distal terminators. The lambda N-function also suppresses termination at or near tR1 in the mutant fusions. N fails to suppress terminators distal to tR1 in the boxA5 fusion, but displays persistent antitermination activity in the boxA16 fusion. A similar reversal of Nun activity occurs when wild-type fusions are introduced into nusA1, nusB5 or nusE71 hosts. We therefore suggest that Nun and N can interact with RNA polymerase in the absence of wild-type boxA, nusA, nusB or nusE, but that the complex formed with mutant components differs functionally from wild-type.

Structure and function of the nun gene and the immunity region of the lambdoid phage HK022.

The immunity region of the lambdoid phage, HK022, has been sequenced. The HK022 repressor gene, its cognate operators and promoters, and several early phage genes can be discerned. The overall design of the immunity region resembles that of other lambdoid phages. The location of the HK022 nun gene, whose product excludes superinfecting lambda by terminating transcription at (or near) the lambda nut sites, is analogous to that of gene N in lambda. nun is preceded by sequences similar to the lambda nut sites and the lambda pL promoter and is followed by several transcription termination signals. Despite these similarities, Nun is required neither for the lytic nor the lysogenic pathway of phage development. Again, unlike N, Nun is expressed in a prophage, perhaps from a promoter other than pL. We suggest that Nun and N have diverged in evolution and now perform different functions for their respective phages. Although Nun and N compete at the lambda nut sites and interact with the same host Nus proteins, they are only distantly related in predicted amino acid sequence. The presence of transcription terminators in the pL operon suggests that the expression of the HK022 early functions, like those of lambda, entails an antitermination mechanism. However, Nun does not appear to be an essential component of this mechanism. Our most economic model is that the HK022 nutL sequence suppresses pL operon terminators in the absence of a phage-encoded antitermination protein. Striking homologies between the HK022 nutL sequence and related sequences in the Escherichia coli rrn operons support this notion. Alternatively, a phage antitermination gene may be located outside the pL operon.

An Escherichia coli mutant unable to support site-specific recombination of bacteriophage lambda.

We report the isolation of mutations in, and the characterization of, an Escherichia coli gene, hip, that is required for site-specific recombination of phage lambda. hip mutants are recessive and are located near minute 20 on the linkage map. The gene product is not vital to bacterial growth, since deletion mutants are viable. The absence of hip product reduces lambda integration to barely detectable levels and also reduces prophage excision, but less drastically. Certain mutations in the lambda int gene partially restore integration and excision in hip- hosts. Homologous recombination promoted by recA does not require hip function. In addition to their defect in site-specific recombination, hip mutants are unable to support lytic growth of phage Mu or of certain lambda mutants. Their pleiotropic phenotype closely resembles that of himA mutants, but complementation, mapping and DNA sequencing show that hip and himA are different genes.

Antitermination of early transcription in phage HK022. Absence of a phage-encoded antitermination factor.

In phage lambda and its relatives most early phage genes are located downstream from transcription termination sites, and full gene expression requires suppression of termination (or antitermination). Phage HK022, a lambda relative, also antiterminates early transcription, but, unlike its relatives, does so in the absence of any active phage gene product. We found no functional equivalent of the lambda N antitermination protein in HK022. In addition, nus mutations, which alter host proteins required for lambda antitermination, have no apparent effect on HK022 early gene expression. We have shown that terminators located several thousand base-pairs from the start point of transcription are suppressed, and that in the left operon suppression requires a short, promoter-proximal segment. A 40 bp region within this segment is repeated in the right operon. The chromosomal locations of these repeated segments resemble those of the nut antitermination sites of other lambdoid phages, but the HK022 sites lack the conserved sequence elements of the nut sites. It appears that HK022 antiterminates early transcription in a novel way.

Gene 3 endonuclease of bacteriophage T7 resolves conformationally branched structures in double-stranded DNA.

Gene 3 endonuclease of bacteriophage T7 has been expressed from the cloned gene, purified, and characterized as to its activity on different DNA substrates. Besides its known strong preference for cutting single-stranded DNA rather than double-stranded DNA, the enzyme has a strong preference for cutting conformationally branched structures in double-stranded DNA, either X or Y-shaped branches. Three types of branched DNA substrates were used: relaxed circular DNAs containing large cruciform structures (a model for Holliday structures, presumed intermediates in genetic recombination); X-shaped molecules having a limited potential for branch migration, made from the cloned phage and bacterial arms of the lambda attachment site; and Y-shaped molecules, made by hybridizing molecules homologous except for a 2 X 21 base-pair palindrome in one of them. Gene 3 endonuclease cuts two opposing strands at or near the branchpoint to resolve these substrates into linear molecules, and does not cut the potentially single-stranded tips of the stem-and-loop structure generated from the palindrome. The position of the cleavage points on the equivalent arm of two X-shaped molecules, constructed from wild-type and mutant lambda attachment sites, show that the enzyme can cut at several different sites within or slightly 5' of the limited region of branch migration. The various activities of gene 3 endonuclease are consistent with the known role of this enzyme in genetic recombination, in maturation and packaging of T7 DNA, and in degradation of host DNA, and suggest that the enzyme recognizes a specific structural feature in DNA. Its cleavage specificity, ready availability, and ability to act at physiological pH and ionic conditions may make gene 3 endonuclease useful as a probe for specific DNA structures or for binding of proteins that alter DNA structure.

Role for DNA homology in site-specific recombination. The isolation and characterization of a site affinity mutant of coliphage lambda.

Site-affinity (or saf) mutations change the specificity of prophage insertion. We have isolated a saf mutation of the bacteriophage lambda attachment site by inserting the phage chromosome into and then excising it from a secondary host attachment site. This causes reciprocal exchange of two seven base-pair segments (the overlap regions) that lie within the cores of the two sites. Since the two overlap regions differ from each other in nucleotide sequence, the recombinant sites are mutants. We have determined the effect of overlap region homology on recombination. We found that homology promotes integrative and excisive recombination. This suggests that the two overlap regions interact directly during recombination. The pattern of segregation of the saf mutation during site-specific recombination shows that it lies to the right of the point of genetic exchange about 95% of the time. This is a surprising result because lambda integrative recombination normally occurs by two staggered, reciprocal single-strand exchanges, one at each edge of the overlap region (Mizuuchi et al., 1981). Since saf lies within the overlap region, we might have expected that the point of genetic exchange would occur to the left of saf as often as to the right. We offer two models to account for this. (1) The mutation alters the location of one of the single-strand exchange points. (2) Efficient and strand-specific processing of mismatched base-pairs changes the expected segregation pattern.

The antiterminator RNA of phage HK022.

The put sites of phage HK022 increase the processivity of transcription and thereby promote the expression of viral genes that are located downstream of transcription terminators. RNA polymerase molecules that have traversed a put site are converted to a terminator-resistant form by the put transcript. We analyzed the structure and function of put transcripts by determining the effects of put mutations on terminator read-through, and by probing wild-type and mutant put RNAs with structure-specific nucleases. The results support the prediction that the secondary structure of the active transcript consists of two hairpin stems that are separated by a single unpaired base. The identity of bases in certain bulges and internal loops is important for activity, while that of most bases in the terminal loops is not. Many bases in the stems can be replaced with little or no effect on activity provided that base-pairing is maintained.

Determinants of site-specific recombination in the lambdoid coliphage HK022. An evolutionary change in specificity.

The temperate bacteriophage HK022, like its relative lambda, inserts its chromosome into a specific site in the bacterial chromosome during lysogenization and excises it after induction. However, we find that the recombinational specificities of the two phages differ: they use different bacterial sites, and neither promotes efficient insertion or excision of the other phage chromosome. In order to determine the basis for this difference in specificity, we sequenced the HK022 elements that are involved in insertion and excision, and compared them to the corresponding lambda elements. The location, orientation, size and overall arrangement of the int and xis genes and the phage attachment sites are nearly identical in the two genomes, as is common for other functionally related elements in lambdoid phages. The Xis proteins of the two phages are functionally interchangeable, and their predicted amino acid sequences differ by but one residue. In contrast, the two Int proteins are not functionally interchangeable, and their sequences, although similar, differ at many positions. These sequence differences are not uniformly distributed: the amino-terminal 55 residues are completely conserved, but the remaining 302 show a pattern of differences interspersed with identities and conservative changes. These findings imply that the specificity difference between HK022 and lambda site-specific recombination is a consequence of the inability of the respective Int proteins to recognize pairs of heterologous attachment sites. The two phage attachment sites are remarkably similar, especially the two "arm" segments, which in lambda contain binding sites for Int, Xis and integration host factor. They are less similar in the segment between the two arms, which in lambda contains the points of recombinational strand exchange and a second class of binding site for Int protein (the "core-type" sites). The two bacterial attachment sites are quite different, although both have a short stretch of perfect homology with their respective phage partners at the points of strand exchange. We propose that the two Int proteins recognize similar or identical sites in the arms of their cognate attachment sites, and that differences in binding or action at the core-type sites is responsible for the divergent specificities. Genetic experiments and sequence comparisons suggest that both proteins recognize different but overlapping families of core-type sites, and that divergence in specificity has been achieved by an alternating succession of small, mutually compatible changes in protein and site.

The effect of attachment site mutations on strand exchange in bacteriophage lambda site-specific recombination.

Recombination of phage lambda attachment sites occurs by sequential exchange of the DNA strands at two specific locations. The first exchange produces a Holliday structure, and the second resolves it to recombinant products. Heterology for base substitution mutations in the region between the two strand exchange points (the overlap region) reduces recombination; some mutations inhibit the accumulation of Holliday structures, others inhibit their resolution to recombinant products. To see if heterology also alters the location of the strand exchange points, we determined the segregation pattern of three single and one multiple base pair substitution mutations of the overlap region in crosses with wild type sites. The mutations are known to differ in the severity of their recombination defect and in the stage of strand exchange they affect. The three single mutations behaved similarly: each segregated into both products of recombination, and the two products of a single crossover were frequently nonreciprocal in the overlap region. In contrast, the multiple mutation preferentially segregated into one of the two recombinant products, and the two products of a single crossover appeared to be fully reciprocal. The simplest explanation of the segregation pattern of the single mutations is that strand exchanges occur at the normal locations to produce recombinants with mismatched base pairs that are frequently repaired. The segregation pattern of the multiple mutation is consistent with the view that both strand exchanges usually occur to one side of the mutant site. We suggest that the segregation pattern of a particular mutation is determined by which stage of strand exchange it inhibits and by the severity of the inhibition.

Bacteriophage lambda int protein may recognize structural features of the attachment sites.

The bacteriophage lambda int protein binds to and promotes polynucleotide strand exchange within specific DNA segments called attachment sites. Previous work strongly suggests that the specificity of int protein action is based, at least in part, on its ability to recognize nucleotide sequences in the attachment sites. We suggest that int protein also recognizes structural features of the attachment sites such as the twist and roll angles between adjacent base pairs. This proposal is based on statistical analysis of the predicted twist and roll angles of a large collection of secondary attachment sites. The analysis shows that the oscillation patterns of these parameters are conserved in regions where int proteins binds.