The genetic dependence of RecBCD-Gam mediated double strand end repair in Escherichia coli

The repair of double strand breaks after gamma-irradiation in wild-type Escherichia coli lysogenic for lambda cI857 red3 is more efficient when lambda Gam protein is present. This phenomenon, called gam dependent radioresistance, requires the interaction of RecBCD enzyme and Gam protein. We compared cell survival after gamma-irradiation in wild-type and mutant lysogens with and without induction of Gam by transient heat treatment of the cells (6 min, 42 degrees C). The main conclusions are: (1) the RecBCD-Gam pathway of recombination repair is similar but not equivalent to RecBCD, a pathway operating in recD mutants; (2) the RecBCD-Gam pathway is dependent on recJ, recQ and recN gene products and it is proposed that the RecBCD-Gam complex has ability to load RecA protein onto single strand DNA.     

The role of DNA recombination in herpes simplex virus DNA replication

In many organisms the processes of DNA replication and recombination are closely linked. For instance, in bacterial and eukaryotic systems, replication forks can become stalled or damaged, in many cases leading to the formation of double stranded breaks. Replication restart is an essential mechanism in which the recombination and repair machinery can be used to continue replication after such a catastrophic event. DNA viruses of bacteria such as lambda and T4 also rely heavily on DNA recombination to replicate their genomes and both viruses encode specialized gene products which are required for recombination-dependent replication. In this review, we examine the linkage between replication and recombination in the eukaryotic pathogen, Herpes Simplex Virus Type 1 (HSV-1). The evidence that recombination plays an intrinsic role in HSV-1 DNA replication and the infection process will be reviewed. We have recently demonstrated that HSV-1 encodes two proteins which may be analogous to the lambda phage recombination system, Red(alpha) and beta. The HSV-1 alkaline nuclease, a 5' to 3' exonuclease, and ICP8, a single stranded DNA binding protein, can carry out strand annealing reactions similar to those carried out by the lambda Red system. In addition, evidence suggesting that host recombination proteins may also be important for HSV-1 replication will be reviewed. In summary, it is likely that HSV-1 infection will require both viral and cellular proteins which participate in various pathways of recombination and that recombination-dependent replication is essential for the efficient replication of viral genomes.     

Involvement of DNA replication in phage lambda Red-mediated homologous recombination

Crosses between a non-replicating linear bacteriophage lambda chromosome and a replicating plasmid bearing a short cloned segment of lambda DNA were monitored by extracting DNA from infected cells, and analysing it via restriction endonuclease digestion and Southern blots. Recombinant formation resulting from the action of the Red homologous recombination system, observed directly in this way, was found to be fast, efficient, independent of the bacterial recA function and highly dependent upon replication of the target plasmid. These features of the experimental system faithfully model Red-mediated recombination in a lytically infected cell in which phage DNA replication is occurring. Neither of the previously established mechanisms by which the Red system can operate – strand annealing or strand invasion – accounts well for these findings. A third mechanism, replisome invasion, involving replication directly in the recombination mechanism, is invoked as an alternative.     

Complementation of bacteriophage lambda integrase mutants: evidence for an intersubunit active site.

Site-specific recombination of bacteriophage lambda starts with the formation of higher-order protein--DNA complexes, called 'intasomes', and is followed by a series of steps, including the initial DNA cleavage, top-strand exchange, branch migration and bottom-strand exchange, to produce recombinant products. One of the intasomes formed during excisive recombination (the attL complex) is composed of the phage-encoded integrase (Int), integration host factor (IHF) and one of the recombination substrates, attL DNA. Int is the catalytic recombinase and has two different DNA binding domains. When IHF is present, Int binds to two types of sites in attL DNA, the three arm-type sites (P'123) and the core-type sites (B and C') where the reciprocal strand exchange takes place. The Tyr342 residue of Int serves as a nucleophile during strand cleavage and covalently attaches to the DNA through a phosphotyrosyl bond. In vitro complementation assays have been performed for strand cleavage using attL suicide substrates and mutant proteins containing amino acid substitutions at residues conserved in the integrase family of recombinases. We demonstrate that at least two Int monomers are required to form the catalytically-competent species that performs cleavage at the B site. It is likely that the active site is formed by two Int monomers.     

The herpes simplex virus type 1 alkaline nuclease and single-stranded DNA binding protein mediate strand exchange in vitro

The replication of herpes simplex virus type 1 (HSV-1) DNA is associated with a high degree of homologous recombination. While cellular enzymes may take part in mediating this recombination, we present evidence for an HSV-1-encoded recombinase activity. HSV-1 alkaline nuclease, encoded by the UL12 gene, is a 5'-->3' exonuclease that shares homology with Redalpha, commonly known as lambda exonuclease, an exonuclease required for homologous recombination by bacteriophage lambda. The HSV-1 single-stranded DNA binding protein ICP8 is an essential protein for HSV DNA replication and possesses single-stranded DNA annealing activities like the Redbeta synaptase component of the phage lambda recombinase. Here we show that UL12 and ICP8 work together to effect strand exchange much like the Red system of lambda. Purified UL12 protein and ICP8 mediated the complete exchange between a 7.25-kb M13mp18 linear double-stranded DNA molecule and circular single-stranded M13 DNA, forming a gapped circle and a displaced strand as final products. The optimal conditions for strand exchange were 1 mM MgCl(2), 40 mM NaCl, and pH 7.5. Stoichiometric amounts of ICP8 were required, and strand exchange did not depend on the nature of the double-stranded end. Nuclease-defective UL12 could not support this reaction. These data suggest that diverse DNA viruses appear to utilize an evolutionarily conserved recombination mechanism.     

Bacteriophage P22 Abc2 protein binds to RecC increases the 5' strand nicking activity of RecBCD and together with lambda bet, promotes Chi-independent recombination

Bacteriophage P22 Abc2 protein binds to the RecBCD enzyme from Escherichia coli to promote phage growth and recombination. Overproduction of the RecC subunit in vivo, but not RecB or RecD, interfered with Abc2-induced UV sensitization, revealing that RecC is the target for Abc2 in vivo. UV-induced ATP crosslinking experiments revealed that Abc2 protein does not interfere with the binding of ATP to either the RecB or RecD subunits in the absence of DNA, though it partially inhibits RecBCD ATPase activity. Productive growth of phage P22 in wild-type Salmonella typhimurium correlates with the presence of Abc2, but is independent of the absolute level of ATP-dependent nuclease activity, suggesting a qualitative change in the nature of Abc2-modified RecBCD nuclease activity relative to the native enzyme. In lambda phage crosses, Abc2-modified RecBCD could substitute for lambda exonuclease in Red-promoted recombination; lambda Gam could not. In exonuclease assays designed to examine the polarity of digestion, Abc2 protein qualitatively changes the nature of RecBCD double-stranded DNA exonuclease by increasing the rate of digestion of the 5' strand. In this respect, Abc2-modified RecBCD resembles a RecBCD molecule that has encountered the recombination hotspot Chi. However, unlike Chi-modified RecBCD, Abc2-modified RecBCD still possesses 3' exonuclease activity. These results are discussed in terms of a model in which Abc2 converts the RecBCD exonuclease for use in the P22 phage recombination pathway. This mechanism of P22-mediated recombination distinguishes it from phage lambda recombination, in which the phage recombination system (Red) and its anti-RecBCD function (Gam) work independently.     

A novel structure of DNA repair protein RecO from Deinococcus radiodurans

Recovery of arrested replication requires coordinated action of DNA repair, replication, and recombination machineries. Bacterial RecO protein is a member of RecF recombination repair pathway important for replication recovery. RecO possesses two distinct activities in vitro, closely resembling those of eukaryotic protein Rad52: DNA annealing and RecA-mediated DNA recombination. Here we present the crystal structure of the RecO protein from the extremely radiation resistant bacteria Deinococcus radiodurans (DrRecO) and characterize its DNA binding and strand annealing properties. The RecO structure is totally different from the Rad52 structure. DrRecO is comprised of three structural domains: an N-terminal domain which adopts an OB-fold, a novel alpha-helical domain, and an unusual zinc-binding domain. Sequence alignments suggest that the multidomain architecture is conserved between RecO proteins from other bacterial species and is suitable to elucidate sites of protein-protein and DNA-protein interactions necessary for RecO functions during the replication recovery and DNA repair.     

DNA arms do the legwork to ensure the directionality of lambda site-specific recombination

The integrase protein of bacteriophage lambda (Int) catalyzes site-specific recombination between lambda phage and Escherichia coli genomes. Int is a tyrosine recombinase that binds to DNA core sites via a C-terminal catalytic domain and to a collection of arm DNA sites, distant from the site of recombination, via its N-terminal domain. The arm sites, in conjunction with accessory DNA-bending proteins, provide a means of regulating the efficiency and directionality of Int-catalyzed recombination. Recent crystal structures of lambda Int tetramers bound to synaptic and Holliday junction intermediates, together with new biochemical data, suggest a mechanism for the allosteric control of the recombination reaction through arm DNA binding interactions.     

The Holliday junction intermediates of lambda integrative and excisive recombination respond differently to the bending proteins integration host factor and excisionase.

In lambda site-specific recombination, the integrative and excisive reactions proceed via two different Holliday junction intermediates, both of which are generated and resolved by a pair of sequentially ordered single strand exchanges. Factors affecting the directionality and efficiency of the second pair of strand exchanges were examined using artificial Holliday junctions (chi-forms). The integrative and excisive recombination intermediates respond differently to the accessory DNA bending proteins integration host factor and excisionase (Xis). These differences between the two recombination intermediates result from a different interaction pattern between proteins binding to the left (P arm) and right (P' arm) of the crossover region. The effect of Xis protein on the directionality of resolution, i.e. the choice of which strands are exchanged, is consistent with a role in promoting the second strand exchange during excision. Proteins binding to the left of the crossover region (P arm) primarily influence the directionality of resolution, while proteins binding to the right (P' arm) have a greater effect on the overall efficiency of resolution. Together, the effect of proteins binding to sites in the P and P' arms is to greatly enhance resolution of the two different Holliday intermediates and to favor resolution in the 'forward' direction for both integrative and excisive recombination.     

The homologous recombination system of phage lambda. Pairing activities of beta protein

The red genes of phage lambda specify two proteins, exonuclease and beta protein, which are essential for its general genetic recombination in recA- cells. These proteins seem to occur in vivo as an equimolar complex. In addition, beta protein forms a complex with another polypeptide, probably of phage origin, of Mr 70,000. The 70-kDa protein appears to be neither a precursor nor an aggregated form of either exonuclease or beta protein, since antibodies directed against the latter two proteins failed to react with 70-kDa protein on Ouchterlony double diffusion analysis. beta protein promotes Mg2+-dependent renaturation of complementary strands (Kmiec, E., and Holloman, W. K. (1981) J. Biol. Chem. 256, 12636-12639). To look for other pairing activities of beta protein, we developed methods of purification to free it of associated exonuclease. Exonuclease-free beta protein appeared unable to cause the pairing of a single strand with duplex DNA; however, like Escherichia coli single strand binding protein (SSB), beta protein stimulated formation of joint molecules by recA protein from linear duplex DNA and homologous circular single strands. Like recA protein, but unlike SSB, beta protein promoted the joining of the complementary single-stranded ends of phage lambda DNA. beta protein specifically protected single-stranded DNA from digestion by pancreatic DNase. The half-time for renaturation catalyzed by beta protein was independent of DNA concentration, unlike renaturation promoted by SSB and spontaneous renaturation, which are second order reactions. Thus, beta protein resembles recA protein in its ability to bring single-stranded DNA molecules together and resembles SSB in its ability to reduce secondary structure in single-stranded DNA.     

Identification of two functional regions in Fis: the N-terminus is required to promote Hin-mediated DNA inversion but not lambda excision.

The Fis protein of E. coli binds to a recombinational enhancer sequence that is required to stimulate Hin-mediated DNA inversion. Fis is also required for efficient lambda prophase excision in vivo. The properties of mutant Fis proteins were examined in vivo and in vitro with respect to their stimulatory effects on these two different site-specific DNA recombination reactions. Both recombination reactions are dramatically affected by mutations altering a helix-turn-helix DNA binding motif located near the Fis C-terminus (residues 74-93). These mutations invariably decrease DNA binding affinity and some cause reduced DNA bending. Mutations in the Fis N-terminal region reduce or abolish the stimulation of Hin-mediated DNA recombination by Fis, but have little or no effect on DNA binding or lambda excision. We conclude that there are at least two functionally distinct domains in Fis: a C-terminal DNA binding region that is required for promoting both DNA recombination reactions and an N-terminal region that is uniquely required for Hin-mediated inversion.     

Terminase host factor: a histone-like E. coli protein which can bind to the cos region of bacteriophage lambda DNA.

Terminase Host Factor (THF), an E. coli protein capable of fulfilling the host factor requirement for in vitro bacteriophage lambda terminase activity, displays properties characteristic of the prokaryotic type II DNA-binding or "histone-like" proteins. It is a 22 K basic, heat- and acid-stable protein which binds non-specifically to various DNAs. Conditions can be established, however, where THF binds preferentially to the cohesive end site (cos) of lambda DNA forming several distinct complexes as visualized by band retardation in polyacrylamide gels. DNase I footprinting reveals that THF can protect several regions of the top strand on the right side (+) of cos but does not bind as well to the left side (-). The binding regions are separated either by unprotected or by DNase I- hypersensitive bases. Under the conditions used in these experiments, DNA which does not contain cos lambda sequences does not show this pattern of protection. Several repeated motifs in the cos lambda nucleotide sequence may represent a consensus sequence for THF interaction. THF may be similar to other "histone-like" proteins which display both non-specific and selective DNA-binding capacities.     

Rad51 protein involved in repair and recombination in S. cerevisiae is a RecA-like protein.

The RAD51 gene of S. cerevisiae is involved in mitotic recombination and repair of DNA damage and also in meiosis. We show that the rad51 null mutant accumulates meiosis-specific double-strand breaks (DSBs) at a recombination hotspot and reduces the formation of physical recombinants. Rad51 protein shows structural similarity to RecA protein, the bacterial strand exchange protein. Furthermore, we have found that Rad51 protein is similar to RecA in its DNA binding properties and binds directly to Rad52 protein, which also plays a crucial role in recombination. These results suggest that the Rad51 protein, probably together with Rad52 protein, is involved in a step to convert DSBs to the next intermediate in recombination. Rad51 protein is also homologous to a meiosis-specific Dmc1 protein of S. cerevisiae.     

Site-specific photo-cross-linking between lambda integrase and its DNA recombination target

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and is composed of three domains as follows: an amino-terminal domain that binds with high affinity to "arm-type" sequences within the recombination target DNA (att sites), a carboxyl-terminal domain that contains all of the catalytic functions, and a central domain that contributes significantly to DNA binding at the "core-type" sequences where DNA cleavage and ligation are executed. We constructed a family of core-type DNA oligonucleotides, each of which contained the photoreactive analog 4-thiodeoxythymidine (4-thioT) at a different position. When tested for their respective abilities to promote covalent cross-links with Int after irradiation with UV light at 366 nm, one oligonucleotide stood out dramatically. The 4-thioT substitution on the DNA strand opposite the site of Int cleavage led to photo-induced cross-linking efficiencies of approximately 20%. The efficiency and specificity of Int binding and cleavage at this 4-thioT-substituted core site was shown to be largely uncompromised, and its ability to participate in a full site-specific recombination reaction was reduced only slightly. Identification of the photo-cross-linked residue as Lys-141 in the central domain provides, along with other results, several insights about the nature of core-type DNA recognition by the bivalent recombinases of the lambda Int family.     

Biological roles of the Escherichia coli RuvA, RuvB and RuvC proteins revealed

In Escherichia coli, the ruvA, ruvB and ruvC gene products are required for genetic recombination and the recombinational repair of DNA damage. New studies suggest that these three proteins function late in recombination and process Holliday junctions made by RecA protein-mediated strand exchange. In vitro, RuvA protein binds a Holliday junction with high affinity and, together with RuvB (an ATPase), promotes ATP-dependent branch migration of the junction leading to the formation of heteroduplex DNA. The third protein, RuvC, which acts independently of RuvA and RuvB, resolves recombination intermediates by specific endonucleolytic cleavage of the Holliday junction.     

The bacteriophage lambda O and P protein initiators promote the replication of single-stranded DNA.

A soluble enzyme system that specifically initiates lambda dv plasmid DNA replication at a bacteriophage lambda replication origin [Wold et al. (1982) Proc. Natl. Acad. Sci. USA 79, 6176-6180] is also capable of replicating the single-stranded circular chromosomes of phages M13 and phi X174 to a duplex form. This chain initiation on single-stranded templates is novel in that it is absolutely dependent on the lambda O and P protein chromosomal initiators and on several Escherichia coli proteins that are known to function in the replication of the lambda chromosome in vivo, including the host dnaB, dnaG (primase), dnaJ and dnaK replication proteins. Strand initiation occurs at multiple sites following an O and P protein-dependent pre-priming step in which the DNA is converted into an activated nucleoprotein complex containing the bacterial dnaB protein. We propose a scheme for the initiation of DNA synthesis on single-stranded templates in this enzyme system that may be relevant to strand initiation events that occur during replication of phage lambda in vivo.     

Specific DNA cleavage mediated by the integrase of conjugative transposon Tn916.

The conjugative transposon Tn916 encodes a protein called INT(Tn916) which, based on DNA sequence comparisons, is a member of the integrase family of site-specific recombinases. Integrase proteins such as INT(lambda), FLP, and XERC/D that promote site-specific recombination use characteristic, conserved amino acid residues to catalyze the cleavage and ligation of DNA substrates during recombination. The reaction proceeds by a two-step transesterification reaction requiring the formation of a covalent protein-DNA intermediate. Different requirements for homology between recombining DNA sites during integrase-mediated site-specific recombination and Tn916 transposition suggest that INT(Tn916) may use a reaction mechanism different from that used by other integrase recombinases. We show that purified INT(Tn916) mediates specific cleavage of duplex DNA substrates containing the Tn916 transposon ends and adjacent bacterial sequences. Staggered cleavages occur at both ends of the transposon, resulting in 5' hydroxyl protruding ends containing coupling sequences. These are sequences that are transferred with the transposon from donor to recipient during conjugative transposition. The nature of the cleavage products suggests that a covalent protein-DNA linkage occurs via a residue of INT(Tn916) and the 3'-phosphate group of the DNA. INT(Tn916) alone is capable of executing the strand cleavage step required for recombination during Tn916 transposition, and this reaction probably occurs by a mechanism similar to that of other integrase family site-specific recombinases.     

Involvement of Escherichia coli DNA Replication Proteins in Phage Lambda Red-Mediated Homologous Recombination

The Red recombination system of bacteriophage lambda is widely used for genetic engineering because of its ability to promote recombination between bacterial chromosomes or plasmids and linear DNA species introduced by electroporation. The process is known to be intimately tied to replication, but the cellular functions which participate with Red in this process are largely unknown. Here two such functions are identified: the GrpE-DnaK-DnaJ chaperone system, and DNA polymerase I. Mutations in either function are found to decrease the efficiency of Red recombination. grpE and dnaJ mutations which greatly decrease Red recombination with electroporated DNA species have only small effects on Red-mediated transduction. This recombination event specificity suggests that the involvement of GrpE-DnaJ-DnaK is not simply an effect on Red structure or stability.     

Site-specific recombination intermediates trapped with suicide substrates

A family of novel substrates was designed to enable the efficient accumulation of intermediates in site-specific recombination. Strategically placed nicks allow these "suicide substrates" to initiate the reaction but prevent its completion or reversal. Consequently, it has been possible to determine that lambda site-specific recombination proceeds by a pair of sequential single-strand exchanges. These results rule out that class of models invoking a concerted cutting of all four DNA strands. The sequential strand exchanges are executed in a strictly prescribed order that is the same in both integrative and excisive recombination. This specified order appears to be governed by the arrangement of bound proteins distal to the sites of strand exchange. Furthermore, when provided with an appropriate 5' OH acceptor, the Integrase protein has the capacity to execute a single DNA strand transfer in a nonreciprocal reaction.