Unexpected structural diversity in DNA recombination: the restriction endonuclease connection

Transposition requires a coordinated series of DNA breakage and joining reactions. The Tn7 transposase contains two proteins: TnsA, which carries out DNA breakage at the 5' ends of the transposon, and TnsB, which carries out breakage and joining at the 3' ends of the transposon. TnsB is a member of the retroviral integrase superfamily whose hallmark is a conserved DDE motif. We report here the structure of TnsA at 2.4 A resolution. Surprisingly, the TnsA fold is that of a type II restriction endonuclease. Thus, Tn7 transposition involves a collaboration between polypeptides, one containing a DDE motif and one that does not. This result indicates that the range of biological processes that utilize restriction enzyme-like folds also includes DNA transposition.     

On the mechanism of mitotic recombination in Aspergillus nidulans. I. Intragenic recombination and DNA replication

Summary Adenine or pABA starvation induce mitotic recombination within the ad9 and paba1 cistrons respectively. Adenine concentrations in the plating medium as low as 1×10^-8 M increase recombination frequency; the concentration optimal in respect to induced recombination frequency is 5×10^-7 M.Recombination within the paba1 cistron is stimulated by low pABA concentrations, or caseine hydrolysate, or methionine.Aminopterin applied for one or two hours before conidia of pABA-requiring diploid are plated on proper selective media, induces recombination within the pro1, ad9 and paba1 cistrons. Conclusion is drawn that it is adenine or thymine starvation which induce mitotic recombination.The implications of this and other similar evidence are discussed.     

Illegitimate replication of linear hepadnavirus DNA through nonhomologous recombination.

Linear hepadnavirus DNA in primary hepatocyte cultures efficiently participates in intra- and intermolecular nonhomologous recombination at its ends. The products of this recombination are (i) monomeric covalently closed circular DNAs (cccDNAs) with deletions and insertions around the site of joining and (ii) oligomeric forms in which monomers are joined near the ends in random orientation. A fraction of monomeric cccDNAs can serve as intermediates in further DNA replication through at least five generations of nonhomologous recombination in a process we call illegitimate replication. We suggest that the monomeric and oligomeric linear DNAs produced by illegitimate replication may be precursors of the integrated and other high-molecular-weight hepadnaviral DNA forms seen in chronic infection.     

Role of recombination occurring during DNA replication

Unequal crossing over between direct DNA repeats of sister chromosomes occurs during DNA replication in Escherichia coli. Such exchanges yield tandem duplications and thereby increase the expression of the genes involved. Nonhomologous cohesion of sister chromosomes and unequal crossing over were assumed to take place when the replication fork stops. When the replication forks moves continuously, homologous exchanges between sister chromosomes ensure their postreplication repair.     

Chromatin replication: Finding the right connection.

Nucleosomes are preferentially assembled on replicating DNA by chromatin assembly factor 1; recent studies have shown that replicated DNA is marked for assembly into chromatin by the replication-fork-associated protein PCNA.     

Early intermediates in bacteriophage T4 DNA replication and recombination.

We investigated, by density gradients and subsequent electron microscopy, vegetative T4 DNA after single or multiple infection of Escherichia coli with wild-type T4. Our results can be summarized as follows. (i) After single infection (i.e., when early intermolecular recombination could not occur), most, if not all, T4 DNA molecules initiated the first round of replication with a single loop. (ii) After multiple infection, recombinational intermediates containing label from both parents first appeared as early as 1 min after the onset of replication, long before all parental DNA molecules had finished their first round and before secondary replication was detectable. (iii) At the same time, in multiple infections only, complex, highly branched concatemeric T4 DNA first appeared. (iv) Molecules in which two loops or several branches were arranged in tandem were only found after multiple infections. (v) Secondary loops within primary loops were seen after both single and multiple infections, but they were rare and many appeared off center. Thus, recombination in wild-type T4-infected cells occurred very early, and the generation of multiple tandem loops or branches in vegetative T4 DNA depended on recombination. These results are consistent with the previous finding (A. Luder and G. Mosig, Proc. Natl. Acad. Sci. U.S.A. 79:1101-1105, 1982) that most secondary growing points of T4 are not initiated from origin sequences but from recombinational intermediates. By these and previous results, the various DNA molecules that we observed are most readily explained as intermediates in DNA replication and recombination according to a model proposed earlier to explain various other aspects of T4 DNA metabolism (Mosig et al., p. 277-295, in D. Ray, ed., The Initiation of DNA Replication, Academic Press, Inc., New York, 1981).     

High-frequency homologous recombination between baculoviruses involves DNA replication

We determined the frequency of DNA recombination between Bombyx mori nucleopolyhedroviruses (BmNPVs) and between BmNPV and the closely related Autographa californica NPV (AcMNPV) in BmN cells, Sf-21 cells, and larvae of Heliothis virescens. The BmN cells were coinfected with two BmNPVs, one with a mutation at the polyhedrin gene (polh) locus and a second carrying a lacZ gene marker cassette. Eleven different BmNPV mutants carrying the lacZ gene marker at various distances (1.4 to 61.7 kb) from polh were used for the coinfections. The Sf-21 cells and larvae of H. virescens were coinfected with wild-type AcMNPV and 1 of the 11 lacZ-marked BmNPV mutants. In BmN cells, high-frequency recombination was detected as early as 15 h postcoinfection but not at 12 h postcoinfection. At 18 h postcoinfection, the mean frequency of recombination ranged between 20.0 and 35.4% when the polh and lacZ marker genes were separated by at least 9.7 kb. When these marker genes were separated by only 1.4 kb, the mean frequency of recombination was 2.7%. In BmN cells, the mean recombination frequency between two BmNPVs increased only marginally when the multiplicity of infection of each virus was increased 10-fold. In Sf-21 cells and the larvae of H. virescens, the recombination frequency between BmNPV and AcMNPV was 相似文献   

Plasmid replication stimulates DNA recombination in Bacillus subtilis

The effects of plasmid replication on the frequency of homologous recombination have been investigated. For that purpose Bacillus subtilis strains that carry in their chromosome directly repeated DNA sequences, and an integrated copy of plasmid pE194 either proximal or distal to the repeats, were constructed. The repeat consists either of 3.9 X 10(3) base pBR322 sequences or 2.1 X 10(3) base B. subtilis chromosomal sequences. As plasmid pE194 is naturally thermosensitive for replication, the activity of the replicon could be regulated. Recombination between the repeated sequences was infrequent (about 10(-4) per generation) when the integrated plasmid did not replicate. It was 20 to 450 times higher when the plasmid was allowed to replicate, provided that the repeats were in the proximity of the plasmid. These results show that plasmid replication stimulates DNA recombination.     

Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication.

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.     

Links between DNA replication and recombination in prokaryotes

Recombination plays a crucial role in underpinning genome duplication, ensuring that replication blocks are removed or bypassed, and that the replication machinery is subsequently reloaded back onto the DNA. Recent studies have identified a surprising variety of ways in which damaged replication forks are repaired and have shown that the mechanism used depends on the nature of the original blocking lesion. Indeed, an emerging theme is that a single recombination enzyme or complex can perform highly varied tasks, depending on the context of the recombination reaction.     

Spontaneous mutagenesis: the roles of DNA repair, replication, and recombination

There appears to be no dearth of mechanisms to explain spontaneous mutagenesis. In the case of base substitutions, data for bacteriophage T4 and especially for E. coli and S. cerevisiae suggest important roles in spontaneous mutagenesis for the error-prone repair of DNA damage (to produce mutations) and for error-free repair of DNA damage (to avoid mutagenesis). Data from the very limited number of studies on the subject suggest that about 50% of the spontaneous base substitutions in E. coli, and perhaps 90% in S. cerevisiae are due to error-prone DNA repair. On the other hand, spontaneous frameshifts and deletions seem to result from mechanisms involving recombination and replication. Spontaneous insertions have been shown to be important in the strongly polar inactivation of certain loci, but it is less important at other loci. Perhaps with continued study, the term "spontaneous mutagenesis" will be replaced by more specific terms such as 5-methylcytosine deamination mutagenesis, fatty acid oxidation mutagenesis, phenylalanine mutagenesis, and imprecise-recombination mutagenesis. While most studies have concentrated on mutator mutations, the most conclusive data for the actual source of spontaneous mutations have come from the study of antimutator mutations. Further study in this area, perhaps along with an understanding of chemical antimutagens, should be invaluable in clarifying the bases of spontaneous mutagenesis.     

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.     

RMPs: recombination/replication mediator proteins.

Enzymes for DNA replication and recombination need to gain access to single-stranded DNA (ssDNA) but ssDNA-binding proteins (SSBs) present an obstacle to the formation of enzyme-ssDNA replication and recombination complexes. A specialized class of SSBs, which we designate as recombination/replication mediator proteins (RMPs), promotes enzyme- ssDNA assembly by overcoming SSB inhibition. RMPs exhibit strong conservation of function across divergent species, and display species-specific interactions with SSB and enzymes to neutralize the SSB barrier to enzyme-ssDNA assembly.     

Paradigms for the three rs: DNA replication, recombination, and repair

The recent decade has engendered a convergence of the otherwise distinct fields of DNA replication, recombination, and repair, as we are learning how these essential transactions can operate in coordination to achieve genomic stability and to ensure cellular viability. In the next decade, we can anticipate a functional understanding of the roles of posttranslational protein modifications in the regulation and prioritizing of pathways for genomic maintenance. The fundamental knowledge gained should lead to more effective clinical intervention in human disease.     

Expansion of DNA repeats in Escherichia coli: effects of recombination and replication functions.

Duplication or expansion of directly repeated sequence elements is associated with a number of human genetic diseases. To study the mechanisms of repeat expansion, we have developed a plasmid assay in Escherichia coli. Our assay involves two simple repeats of 787 bp in length; expansion to three or more copies of the repeat can be selected by restoration of an intact tetracycline-resistance gene. Expansions occurred at relatively high rates, >10(-5), in the population. Both RecA-dependent recombination and RecA-independent slipped misalignments contributed to the observed expansion events. Mutations that impair DNA polymerase III (DnaE, DnaQ subunits) or the replication fork helicase, DnaB, stimulated both RecA-dependent and RecA-independent expansion events. In these respects, the properties of repeat expansion resemble repeat deletion and suggest that difficulties in DNA replication may trigger both classes of rearrangements. About 20% of the RecA-independent expansion events are accompanied by reciprocal sister-chromosome exchange, producing dimeric plasmids carrying one triplicated and one deleted locus. These products are explained by a model involving misaligned strands across the replication fork. This model predicts that the location of a replication stall site may govern the types of resulting rearrangements. The specific location of such a stall site can also, in theory, account for propensity towards expansion or deletion of repeat arrays. This may have relevance to trinucleotide repeat expansion in human genetic disease.     

PriA mediates DNA replication pathway choice at recombination intermediates

We report the reconstitution of the initial steps of the double-strand break-repair pathway where joint molecule formation between a duplex DNA fragment and a circular template by the combined action of RecA, RecBCD, and the single-stranded DNA binding protein provides the substrate for replication fork formation by the restart primosome and the DNA polymerase III holoenzyme. We show that PriA dictates the pathway of replication from the recombination intermediate by inhibiting a nonspecific, strand displacement DNA synthesis reaction and favoring the formation of a bona fide replication fork. Furthermore, we find that RecO and RecR significantly stimulate this recombination-directed DNA replication reaction, and that this stimulation is modulated by the presence of RecF, suggesting that the latter protein may also act as a regulator of the pathway of resolution of the recombination intermediate.     

Theta-type DNA replication stimulates homologous recombination in the Bacillus subtilis chromosome

To test the effects of theta-type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of ∼ 4 kb. The replicon was derived from a broad-host-range plasmid, pAMβ1, which replicates by a unidirectional theta mechanism and is thermosensitive. The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol-resistance gene of plasmid pC194. Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats. The integrated replicon was active at the permissive temperature, and ∼ 25% of the integrated plasmids could be isolated as Y-shaped molecules after restriction, having a branch at the replication origin. Replicon activity stimulated recombination four- to fivefold, as estimated from the proportion of chloramphenicol-sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats. We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.     

Mini- and microsatellite expansions: the recombination connection

It is widely accepted that the large trinucleotide repeat expansions observed in many neurological diseases occur during replication. However, genetic recombination has emerged as a major source of instability for tandem repeats, including minisatellites, and recent studies raise the possibility that it may also be responsible for trinucleotide repeat expansions. We will review data connecting tandem repeat rearrangements and recombination in humans and in eukaryotic model organisms, and discuss the possible role of recombination in trinucleotide repeat expansions in human neurological disorders.     

Herpes simplex virus type 1 recombination: role of DNA replication and viral a sequences.

During the course of infection, elements of the herpes simplex virus type 1 (HSV-1) genome undergo inversion, a process that is believed to occur through the viral a sequences. To investigate the mechanism of this recombinational event, we have developed an assay that detects the deletion of DNA segments flanked by directly repeated a sequences in plasmids transiently maintained in Vero cells. With this assay, we have observed a high frequency of recombination (approximately 8%) in plasmids that undergo replication in HSV-1-infected cells. We also found a low level of recombination between a sequences in plasmids introduced into uninfected cells and in unreplicated plasmids in HSV-1-infected cells. In replicating plasmids, recombination between a sequences occurs at twice the frequency seen with directly repeated copies of a different sequence of similar size. Recombination between a sequences appears to occur at approximately the same time as replication, suggesting that the processes of replication and recombination are closely linked.