T7 Exonuclease (GENE 6) is Necessary for Molecular Recombination of Bacteriophage T7

The role of T7-induced exonuclease (gene 6) in molecular recombination was studied by examining the fate of parental DNA during parental-to-progeny recombination. The method used was to compare infections with T7(+), T7am-6-233 (am gene 6), or T7ts6-136 (ts gene 6) under permissive and nonpermissive conditions. CsCl density gradient analysis of replicative DNA indicated that T7 exonuclease is necessary for recombination to occur, i.e., in the absence of the exonuclease the parental DNA replicated continuously as a hybrid molecule and did not recombine. Further studies under conditions where replicative DNA was denatured and analyzed by CsCl density gradient centrifugation indicated that the exonuclease is also needed for a limited amount of covalent repair of recombinants. A repair function for the T7-induced exonuclease is also suggested by results obtained from alkaline sucrose gradient analysis of replicative DNA. Under conditions nonpermissive for the exonuclease, discontinuities in the DNA accumulated during infection by T7am6-233 or by T7ts6-136.     

Multidimensional analysis of intracellular bacteriophage T7 DNA: effects of amber mutations in genes 3 and 19.

By use of rate-zonal centrifugation, followed by either one- or two-dimensional agarose gel electrophoresis, the forms of intracellular bacteriophage T7 DNA produced by replication, recombination, and packaging have been analyzed. Previous studies had shown that at least some intracellular DNA with sedimentation coefficients between 32S (the S value of mature T7 DNA) and 100S is concatemeric, i.e., linear and longer than mature T7 DNA. The analysis presented here confirmed that most of this DNA is linear, but also revealed a significant amount of circular DNA. The data suggest that these circles are produced during DNA packaging. It is proposed that circles are produced after a capsid has bound two sequential genomes in a concatemer. The size distribution of the linear, concatemeric DNA had peaks at the positions of dimeric and trimeric concatemers. Restriction endonuclease analysis revealed that most of the mature T7 DNA subunits of concatemers were joined left end to right end. However, these data also suggest that a comparatively small amount of left-end to left-end joining occurs, possibly by blunt-end ligation. A replicating form of T7 DNA that had an S value greater than 100 (100S+ DNA) was also found to contain concatemers. However, some of the 100S+ DNA, probably the most branched component, remained associated with the origin after agarose gel electrophoresis. It has been found that T7 protein 19, known to be required for DNA packaging, was also required to prevent loss, probably by nucleolytic degradation, of the right end of all forms of intracellular T7 DNA. T7 gene 3 endonuclease, whose activity is required for both recombination of T7 DNA and degradation of host DNA, was required for the formation of the 32S to 100S molecules that behaved as concatemers during gel electrophoresis. In the absence of gene 3 endonuclease, the primary accumulation product was origin-associated 100S+ DNA with properties that suggest the accumulation of branches, primarily at the left end of mature DNA subunits within the 100S+ DNA.     

Role of gene 6 exonuclease in the replication and packaging of bacteriophage T7 DNA

When bacteriophage T7 gene 6 exonuclease is genetically removed from T7-infected cells, degradation of intracellular T7 DNA is observed. By use of rate zonal centrifugation, followed by either pulsed-field agarose gel electrophoresis or restriction endonuclease analysis, in the present study, the following observations were made. (1) Most degradation of intracellular DNA requires the presence of T7 gene 3 endonuclease and is independent of DNA packaging; rapidly sedimenting, branched DNA accumulates when both the gene 3 and gene 6 products are absent. (2) A comparatively small amount of degradation requires packaging and occurs at both the joint between genomes in a concatemer and near the left end of intracellular DNA; DNA packaging is only partially blocked and end-to-end joining of genomes is not blocked in the absence of gene 6 exonuclease. (3) Fragments produced in the absence of gene 6 exonuclease are linear and do not further degrade; precursors of the fragments are non-linear. (4) Some, but not most, of the cleavages that produce these fragments occur selectively near two known origins of DNA replication. On the basis of these observations, the conclusion is drawn that most degradation that occurs in the absence of T7 gene 6 exonuclease is caused by cleavage at branches. The following hypothesis is presented: most, possibly all, of the extra branching induced by removal of gene 6 exonuclease is caused by strand displacement DNA synthesis at the site of RNA primers of DNA synthesis; the RNA primers, produced by multiple initiations of DNA replication, are removed by the RNase H activity of gene 6 exonuclease during a wild-type T7 infection. Observation of joining of genomes in the absence of gene 6 exonuclease and additional observations indicate that single-stranded terminal repeats required for concatamerization are produced by DNA replication. The observed selective shortening of the left end indicates that gene 6 exonuclease is required for formation of most, possibly all, mature left ends.     

Bacteriophage T7 DNA packaging. III. A "hairpin" end formed on T7 concatemers may be an intermediate in the processing reaction

An unusual left end (M-end) has been identified on bacteriophage T7 DNA isolated from T7-infected cells. This end has a "hairpin" structure and is formed at a short inverted repeat sequence centered around nucleotide 39,587 of T7, 190 base-pairs to the left of the site where a mature left end is formed on the T7 concatemer. We do not detect the companion right end that would be formed if the M-end is produced by a double-stranded cut on the T7 concatemer. This suggests that the hairpin left end may be generated from a single-stranded cut in the DNA that is used to prime rightward DNA synthesis. The formation of M-end does not require the products of T7 genes 10, 18 or 19, proteins that are essential for the formation of mature T7 ends. During infection with a T7 gene 3 (endonuclease) mutant, phage DNA synthesis is reduced and the concatemers are not processed into unit length DNA molecules, but both M-end and the mature right end are formed on the concatemer DNA. These two ends are also found associated with the large, rapidly sedimenting concatemers formed during a normal T7 infection while the mature left end is present only on unit length T7 DNA molecules. We propose that DNA replication primed from the hairpin end produced by a nick in the inverted repeat sequence provides a mechanism to duplicate the terminal repeat before DNA packaging. Packaging is initiated with the formation of a mature right end on the branched concatemer and, as the phage head is filled, the T7 gene 3 endonuclease may be required to trim the replication forks from the DNA. Concatemer processing is completed by the removal of the 190 base-pair hairpin end to produce the mature left end.     

Studies on an Endonuclease Activity Associated with the Bacteriophage T7 DNA-Membrane Complex

Infection of Escherichia coli with bacteriophage T7 results in the formation of an endonuclease which is selectively associated with the T7 DNA-membrane complex. A specificity of association with the complex is indicated by the finding that the enzyme is completely resolved from a previously described T7 endonuclease I. When membrane complexes containing (3)H-labeled in vivo synthesized DNA are incubated in the standard reaction mixture a specific cleavage product is formed which is about one-fourth the size of T7 DNA. The endonuclease associated with the complex produces a similar cleavage product after extensive incubation with native T7 DNA or T7 concatemers. Degradation of concatemers occurs by a mechanism in which the DNA is converted to molecules one-half the size of T7. This product is in turn converted to fragments one-fourth the size of mature phage DNA. The endonuclease is not present in membrane complexes from uninfected cells or cells infected with gene 1 mutants. The enzyme activity is, however, present in cells infected with mutants defective in T7 DNA synthesis or maturation.     

In vitro packaging of bacteriophate T7 DNA synthesized in vitro.

An in vitro DNA packaging system was used to encapsulate T7 DNA that had been synthesized by extracts prepared from gently lysed Escherchia coli infected with bacteriophage T7 carrying amber mutations in gene 3 or in both genes 3 and 6. Isopycnic centrifugation of density-labeled wild-type DNA was employed in an effort to separate product from template; suppressor-free indicator bacteria were used to eliminate contributions from endogenous DNA or contaminating phage. Additional controls indicated that fragmented DNA is packaged in vitro only with very low efficiency and that the frequency of recombination during packaging is too low to affect interpretation of these experiments. T7 DNA replicated by extracts prepared using T7 mutants deficient in both genes 3 and 6 could be packaged in vitro with an efficiency comparable to that found when highly purified virion T7 DNA was used. When T7 deficient in the gene 3 endonuclease but with normal levels of the gene 6 exonuclease was used, fast-sedimentingconcatemer-like DNA structures were formed during in vitro DNA synthesis. Electron microscopy revealed many branched and highly complex DNA structures formed during this reaction. This concatemer-like DNA was encapsulated in vitro with an efficiency significantly greater than that found for DNA the length of a single T7 genome.     

Flap Endonuclease Activity of Gene 6 Exonuclease of Bacteriophage T7

Flap endonucleases remove flap structures generated during DNA replication. Gene 6 protein of bacteriophage T7 is a 5′–3′-exonuclease specific for dsDNA. Here we show that gene 6 protein also possesses a structure-specific endonuclease activity similar to known flap endonucleases. The flap endonuclease activity is less active relative to its exonuclease activity. The major cleavage by the endonuclease activity occurs at a position one nucleotide into the duplex region adjacent to a dsDNA-ssDNA junction. The efficiency of cleavage of the flap decreases with increasing length of the 5′-overhang. A 3′-single-stranded tail arising from the same end of the duplex as the 5′-tail inhibits gene 6 protein flap endonuclease activity. The released flap is not degraded further, but the exonuclease activity then proceeds to hydrolyze the 5′-terminal strand of the duplex. T7 gene 2.5 single-stranded DNA-binding protein stimulates the exonuclease and also the endonuclease activity. This stimulation is attributed to a specific interaction between the two proteins because Escherichia coli single-stranded DNA binding protein does not produce this stimulatory effect. The ability of gene 6 protein to remove 5′-terminal overhangs as well as to remove nucleotides from the 5′-termini enables it to effectively process the 5′-termini of Okazaki fragments before they are ligated.     

RNA-linked nascent DNA pieces in T7 phage-infected Escherichia coli cells. I. Role of gene 6 exonuclease in removal of the linked RNA.

Summary The presence of RNA-linked nascent DNA pieces in T7 phage-infectedEscherichia coli cells has been shown by the selective degradation of the 5-hydroxyl-terminated nascent DNA, produced by alkali or RNase treatment, with spleen exonuclease. At 43°C, the proportion of RNA-linked DNA pieces in nascent short DNA is 50 to 60% in T7ts136 (ts mutant of gene 6) phage-infectedE. coli, whereas that in T7 wild-type phage-infected cells is less than 6%. Joining of the nascent pieces is greatly retarded in T7ts136-infectedE. coli temperature sensitivepolA mutants at 43° C. These results suggest that gene 6 exonuclease plays a role in removal of the linked RNA during the discontinuous replication of T7 DNA.     

Role of the recF gene of Escherichia coli K-12 in lambda recombination

Summary When Escherichia coli K12() lysogens are infected with heteroimmune phage, which are unable to replicate, general recombination between phage and prophage depends on the bacterial recF gene. It has been shown that in E. coli K12 postconjugational recombination, the RecF pathway only works with full efficiency if exonuclease I is absent (Clark 1973). However, results presented in this paper indicate that under conditions in which replication is blocked, the recombination pathway dependant on the recF gene is fully active in producing viral recombinants even, if the phage is Red⁺, in the presence of exonuclease I. In contrast, removal of exonuclease and protein requires elimination of exonuclease I for an efficient RecF pathway. It is concluded that the Red system cooperates with the RecF pathway and that this cooperation involves overcoming the inhibitory effects of exonuclease I. In the absence of exonuclease, protein stimulates recF-dependent recombination but does not suffice to prevent the negative effect of exonuclease I. In the presence of protein, full efficiency of the RecF pathway can be obtained either via cooperation with exonuclease I or, if the viral exonuclease is defective, via inactivation of exonuclease I. Since activity of exonuclease appears necessary to overcome the inhibitory effects of exonuclease I, it is proposed here that exonuclease diverts material from the RecF pathway in a shunt reaction which allows completion of recF-initiated recombinational intermediates via a mechanism insensitive to exonuclease I.When replication is allowed, the Rec system produces viral recombinants mainly via a recF-independent mechanism. However, a major contribution of the RecF pathway to recombination is observed after removal of the Red system and exonuclease I.Obra social de la Caja de Ahorros de Valencia (Director: S. Grisolía)     

Processing of concatemers of bacteriophage T7 DNA in vitro

The T7 chromosome is a double-stranded linear DNA molecule flanked by direct terminal repeats or so-called terminal redundancies. Late in infection bacteriophage T7 DNA accumulates in the form of concatemers, molecules that are comprised of T7 chromosomes joined in a head to tail arrangement through shared terminal redundancies. To elucidate the molecular mechanisms of concatemer processing, we have developed extracts that process concatemeric DNA. The in vitro system consists of an extract of phage T7-infected cells that provides all T7 gene products and minimal levels of endogenous concatemeric DNA. Processing is analyzed using a linear 32P-labeled substrate containing the concatemeric joint. T7 gene products required for in vitro processing can be divided into two groups; one group is essential for concatemer processing, and the other is required for the production of full length left-hand ends. The products of genes 8 (prohead protein), 9 (scaffolding protein), and 19 (DNA maturation) along with gene 18 protein are essential, indicating that capsids are required for processing. In extracts lacking one or more of the products of genes 2 (Escherichia coli RNA polymerase inhibitor), 5 (DNA polymerase), and 6 (exonuclease), full length right-hand ends are produced. However, the left-hand ends produced are truncated, lacking at least 160 base pairs, the length of the terminal redundancy. Gene 3 endonuclease, required for concatemer processing in vivo, is not required in this system. Both the full length left- and right-hand ends produced by the processing reaction are protected from DNase I digestion, suggesting that processing of the concatemeric joint substrate is accompanied by packaging.     

The RAD2 domain of human exonuclease 1 exhibits 5' to 3' exonuclease and flap structure-specific endonuclease activities

The RAD2 family of nucleases includes human XPG (Class I), FEN1 (Class II), and HEX1/hEXO1 (Class III) products gene. These proteins exhibit a blend of substrate specific exo- and endonuclease activities and contribute to repair, recombination, and/or replication. To date, the substrate preferences of the EXO1-like Class III proteins have not been thoroughly defined. We report here that the RAD2 domain of human exonuclease 1 (HEX1-N2) exhibits both a robust 5' to 3' exonuclease activity on single- and double-stranded DNA substrates as well as a flap structure-specific endonuclease activity but does not show specific endonuclease activity at 10-base pair bubble-like structures, G:T mismatches, or uracil residues. Both the 5' to 3' exonuclease and flap endonuclease activities require a divalent metal cofactor, with Mg(2+) being the preferred metal ion. HEX1-N2 is approximately 3-fold less active in Mn(2+)-containing buffers and exhibits <5% activity in the presence of Co(2+), Zn(2+), or Ca(2+). The optimal pH range for the nuclease activities of HEX1-N2 is 7.2-8.2. The specific activity of its 5' to 3' exonuclease function is 2.5-7-fold higher on blunt end and 5'-recessed double-stranded DNA substrates compared with duplex 5'-overhang or single-stranded DNAs. The flap endonuclease activity of HEX1-N2 is similar to that of human flap endonuclease-1, both in terms of turnover efficiency (k(cat)) and site of incision, and is as efficient (k(cat)/K(m)) as its exonuclease function. The nuclease activities of HEX1-N2 described here indicate functions for the EXO1-like proteins in replication, repair, and/or recombination that may overlap with human flap endonuclease-1.     

Genetic recombination of bacteriophage T7 in vivo studied by use of a simple physical assay.

A new physical method was developed to assay genetic recombination of phage T7 in vivo. The assay utilized T7 mutants that carry unique restriction sites and was based on the detection of a new restriction fragment generated by recombination. Using this assay, we reexamined the genetic requirements for recombination of T7 DNA. Our results were in total agreement with previous findings in that recombination required the products of genes 3 (endonuclease), 4 (primase), 5 (DNA polymerase), and 6 (exonuclease). Recombination was found to be independent of DNA ligase and DNA packaging and maturation functions.     

The essential role of bacteriophage T7 endonuclease (gene 3) in molecular recombination

Density transfer and shearing experiments show that the bacteriophage T7 endonuclease (gene 3) is necessary for the dispersion of parental DNA in the newly replicated DNA. These experiments on parental to progeny recombination support previous genetic data (Powling & Knippers, 1974; Kerr & Sadowski, 1975) that the gene 3 protein is essential for T7 recombination. Concatemers containing the newly replicated DNA have been sheared to the size of mature phage DNA and also to quarter molecules. In the presence of gene 3 protein, parental DNA and newly replicated DNA are interspersed. In the absence of gene 3 protein, the parental strand of each sheared DNA molecule is usually found intact.     

Bacteriophage T7 DNA packaging. I. Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection

Bacteriophage T7 DNA is a linear duplex molecule with a 160 base-pair direct repeat (terminal redundancy) at its ends. During replication, large DNA concatemers are formed, which are multimers of the T7 genome linked head to tail through recombination at the terminal redundancy. We define the sequence that results from this recombination, a mature right end joined to the left end of T7 DNA, as the concatemer junction. To study the processing and packaging of T7 concatemers into phage particles, we have cloned the T7 concatemer junction into a plasmid vector. This plasmid is efficiently (at least 15 particles/infected cell) packaged into transducing particles during a T7 infection. These transducing particles can be separated from T7 phage by sedimentation to equilibrium in CsCl. The packaged plasmid DNA is a linear concatemer of about 40 x 10(3) base-pairs with ends at the expected T7 DNA sequences. Thus, the T7 concatemer junction sequence on the plasmid is recognized for processing and packaging by the phage system. We have identified a T7 DNA replication origin near the right end of the T7 genome that is necessary for efficient plasmid packaging. The origin, which is associated with a T7 RNA polymerase promoter, causes amplification of the plasmid DNA during T7 infection. The amplified plasmid DNA sediments very rapidly and contains large concatemers, which are expected to be good substrates for the packaging reaction. When cloned in pBR322, a sequence containing only the mature right end of T7 DNA is sufficient for efficient packaging. Since this sequence does not contain DNA to the right of the site where a mature T7 right end is formed, it was expected that right ends would not form on this DNA. In fact, with this plasmid the right end does not form at the normal T7 sequence but is instead formed within the vector. Apparently, the T7 packaging system can also recognize a site in pBR322 DNA to produce an end for packaging. This site is not recognized solely by a "headful" mechanism, since there can be considerable variation in the amount of DNA packaged (32 x 10(3) to 42 x 10(3) base-pairs). Furthermore, deletion of this region from the vector DNA prevents packaging of the plasmid. The end that is formed in vector DNA is somewhat heterogeneous. About one-third of the ends are at a unique site (nucleotide 1712 of pBR322), which is followed by the sequence 5'-ATCTGT-3'. This sequence is also found adjacent to the cut made in a T7 DNA concatemer to produce a normal T7 right end.     

Yeast Exonuclease 5 Is Essential for Mitochondrial Genome Maintenance

Yeast exonuclease 5 is encoded by the YBR163w (DEM1) gene, and this gene has been renamed EXO5. It is distantly related to the Escherichia coli RecB exonuclease class. Exo5 is localized to the mitochondria, and EXO5 deletions or nuclease-defective EXO5 mutants invariably yield petites, amplifying either the ori3 or ori5 region of the mitochondrial genome. These petites remain unstable and undergo continuous rearrangement. The mitochondrial phenotype of exo5Δ strains suggests an essential role for the enzyme in DNA replication and recombination. No nuclear phenotype associated with EXO5 deletions has been detected. Exo5 is a monomeric 5′ exonuclease that releases dinucleotides as products. It is specific for single-stranded DNA and does not hydrolyze RNA. However, Exo5 has the capacity to slide across 5′ double-stranded DNA or 5′ RNA sequences and resumes cutting two nucleotides downstream of the double-stranded-to-single-stranded junction or RNA-to-DNA junction, respectively.Endonucleases and exonucleases are intimately involved in all aspects of DNA metabolism in the cell. In mitochondria, several constitutive nucleases have been identified that contribute to the proper maintenance of the mitochondrial genome through replication and recombination pathways. In addition, nucleases can localize to mitochondria in response to DNA stress in order to mediate appropriate DNA repair. Among the constitutive mitochondrial nucleases in Saccharomyces cerevisiae are the Nuc1 nuclease that contributes to DNA recombination efficiency and functions in apoptosis (4, 38) and the Cce1 endonuclease that resolves recombination intermediates (29). The Din7 endonuclease is a mitochondrially located 5′ flap endonuclease related to FEN1 (20). While deletion of the gene for either of these enzymes produced marginal mitochondrial phenotypes, more severe phenotypes were observed when combined deletions of these nuclease genes were studied or when they were combined with deletions of other genes involved in DNA recombination or repair, such as MHR1 or MSH1 (20, 22, 27). Recently, human Dna2 was shown to localize to both the nuclear and mitochondrial compartments and to participate in mitochondrial DNA replication and base excision repair (11, 39). Its function in yeast mitochondrial DNA maintenance has not been studied in detail. Finally, the 5′ flap endonuclease FEN1, which normally functions in primer RNA degradation during Okazaki fragment maturation in the nucleus, also localizes to the mitochondrion in response to DNA damage, participating in long-patch base excision repair (19, 23).Since mitochondrial function is not essential to yeast survival, dysfunction caused by mutations of the mitochondrial genome can be readily detected as a loss of respiration function, which is scored as the inability to grow on nonfermentable carbon sources. A defect in the mitochondrial DNA polymerase γ MIP1 results in complete loss of the mitochondrial DNA, and the mutant fails to grow on glycerol-containing media lacking glucose (14). Such cells are designated ρ⁰. Genome maintenance defects can also result in the generation of petite mutants that still contain mitochondrial DNA. Generally, most of the mitochondrial genome has been deleted, and a small origin-containing region has been amplified (ρ⁻). S. cerevisiae contains eight such origin regions that are highly similar in sequence and are distributed over the 86-kb mitochondrial genome (8, 9, 15). Petites that have amplified the ori5 region have been studied more extensively (16, 22).While the nucleases listed above participate in the proper maintenance of the mitochondrial genome through their replication and/or recombination functions, none appears to be essential for the integrity of the mitochondrial genome. One reasonable explanation for these observations is functional redundancy. Indeed, functional nuclease redundancy is quite common; it has been observed in the process of DNA degradation during mismatch repair in Escherichia coli, during Okazaki fragment maturation in yeast, and during the resection of double-stranded breaks in yeast (7, 25, 33). However, the possibility remains that an additional nuclease(s) is active in the mitochondrion. The present paper describes an essential mitochondrial exonuclease that is distantly related to the nuclease domain of RecB, a subunit of the bacterial RecBCD recombinase. This nuclease was discovered over 2 decades ago during a biochemical chromatographic survey of yeast exonucleases and was called exonuclease 5 (3). Initial studies with a partially purified enzyme preparation showed it to be a 5′ exonuclease specific for single-stranded DNA (ssDNA). Here we report the identification of the EXO5 gene and describe comprehensive biochemical and genetic studies that show a critical role for EXO5 in mitochondrial DNA maintenance, presumably through the processing of replication intermediates. Upon deletion of EXO5 or inactivation of its nuclease activity, only ρ⁻ mutants could be recovered. EXO5 has previously been characterized as DEM1 (defects in morphology) because the deletion mutant shows defects in growth and in mitochondrial morphology (10, 12). No nuclear defect associated with an EXO5 deletion has been detected.     

AAV Recombineering with Single Strand Oligonucleotides

Adeno-associated virus (AAV) transduction initiates a signaling cascade that culminates in a transient DNA damage response. During this time, host DNA repair proteins convert the linear single-strand AAV genomes to double-strand circular monomers and concatemers in processes stimulated by the AAV inverted terminal repeats (ITRs). As the orientation of AAV genome concatemerization appears unbiased, the likelihood of concatemerization in a desired orientation is low (less than 1 in 6). Using a novel recombineering method, Oligo-Assisted AAV Genome Recombination (OAGR), this work demonstrates the ability to direct concatemerization specifically to a desired orientation in human cells. This was achieved by a single-strand DNA oligonucleotide (oligo) displaying homology to distinct AAV genomes capable of forming an intermolecular bridge for recombination. This DNA repair process results in concatemers with genomic junctions corresponding to the sequence of oligo homology. Furthermore, OAGR was restricted to single-strand, not duplexed, AAV genomes suggestive of replication-dependent recombination. Consistent with this process, OAGR demonstrated oligo polarity biases in all tested configurations except when a portion of the oligo targeted the ITR. This approach, in addition to being useful for the elucidation of intermolecular homologous recombination, may find eventual relevance for AAV mediated large gene therapy.     

R-loop-dependent rolling-circle replication and a new model for DNA concatemer resolution by mitochondrial plasmid mp1

The mitochondrial (mt) plasmid mp1 of Chenopodium album replicates by a rolling-circle (RC) mechanism initiated at two double-stranded replication origins (dso1 and dso2). Two-dimensional gel electrophoresis and electron microscopy of early mp1 replication intermediates revealed novel spots. Ribonucleotide (R)-loops were identified at dso1, which function as a precursor for the RCs in vivo and in vitro. Bacteriophage T4-like networks of highly branched mp1 concatemers with up to 20 monomer units were mapped and shown to be mainly formed by replicating, invading, recombining and resolving molecules. A new model is proposed in which concatemers were separated into single units by a "snap-back" mechanism and homologous recombination. dso1 is a recombination hotspot, with sequence homology to bacterial Xer recombination cores. mp1 is a unique eukaryotic plasmid that expresses features of phages like T4 and could serve as a model system for replication and maintenance of DNA concatemers.     

Shade avoidance 6 encodes an Arabidopsis flap endonuclease required for maintenance of genome integrity and development

Flap endonuclease-1 (FEN1) belongs to the Rad2 family of structure-specific nucleases. It is required for several DNA metabolic pathways, including DNA replication and DNA damage repair. Here, we have identified a shade avoidance mutant, sav6, which reduces the mRNA splicing efficiency of SAV6. We have demonstrated that SAV6 is an FEN1 homologue that shows double-flap endonuclease and gap-dependent endonuclease activity, but lacks exonuclease activity. sav6 mutants are hypersensitive to DNA damage induced by ultraviolet (UV)-C radiation and reagents that induce double-stranded DNA breaks, but exhibit normal responses to chemicals that block DNA replication. Signalling components that respond to DNA damage are constitutively activated in sav6 mutants. These data indicate that SAV6 is required for DNA damage repair and the maintenance of genome integrity. Mutant sav6 plants also show reduced root apical meristem (RAM) size and defective quiescent centre (QC) development. The expression of SMR7, a cell cycle regulatory gene, and ERF115 and PSK5, regulators of QC division, is increased in sav6 mutants. Their constitutive induction is likely due to the elevated DNA damage responses in sav6 and may lead to defects in the development of the RAM and QC. Therefore, SAV6 assures proper root development through maintenance of genome integrity.