Nick Site Research Articles

A rolling circle replication initiator protein with a nucleotidyl-transferase activity encoded by the plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi.

The plasmid pGT5 from the hyperthermophilic archaeon Pyrococcus abyssi presents similarities to plasmids from the pC194 family that replicate by the rolling circle mechanism. These plasmids encode a replication initiator protein, which activates the replication origin by nicking one of the two DNA strands. The gene encoding the putative Rep protein of pGT5 (Rep75) has been cloned and overexpressed in Escherichia coli, and the recombinant protein has been purified to homogeneity. Rep75 exhibits a highly thermophilic nicking-closing activity in vitro on single-stranded oligonucleotides containing the putative double-stranded replication origin sequence of pGT5. Gel shift analyses on single-stranded oligonucleotides indicate that Rep75 recognizes the single-stranded DNA region upstream of the nicking site via non-covalent interaction and remains covalently linked to the 5'-phosphate of the downstream fragment after nicking. Besides these expected activities, Rep75 contains a dATP (and ATP) terminal transferase activity at the 3'-OH extremity of the nicking site, which had not been reported previously for proteins of this type. Rep75, which is the first replication initiator protein characterized in an archaeon, offers an attractive new model for the study of rolling circle replication.

Conformations of nicked and gapped DNA structures by NMR and molecular dynamic simulations in water.

We have analyzed and compared the molecular structures and dynamics of DNA duplexes containing a nick or a gap of one nucleotide where the base in front of the gap is a guanine. The continuous strand has the sequence 5'(CAGAGTCXCTGGCTC) where the residue X is absent for the nick, 14-mer, and where it is a G residue for the gap. Duplexes were formed with the two corresponding 7-mers. Neither of these is phosphorylated adjacent at the nick site, but it is a good model for a single strand break. For the nick structure, the quantitative NMR data show that the global conformation is very close to canonical B-form DNA, but it displays enhanced local flexibility. For the gap structure, we observe only one species in which the extra G is well stacked into the helix. The two half-helices around this residue also show a B-form conformation. As with the nick duplex, the adjacent G imino protons show enhanced exchange with solvent. The gap does not close completely. Using distance constraints, MD calculations show that the nick conformation is very close to a duplex with no lesion but is indeed more flexible in the central part. The gapped structure shows two families of conformations. One is close to B-DNA, the other is significantly kinked at the gap which reduces the size of the cavity. We observe a spine of hydration within the cavities, similar, but of different geometry in the two cases.

The transfer origin for Bacteroides mobilizable transposon Tn4555 is related to a plasmid family from gram-positive bacteria.

Conjugal transfer of Bacteroides mobilizable transposon Tn4555 was examined with an Escherichia coli-based assay system. It was shown that mobilization required the cis-acting oriT(Tn) region and that the Tn4555 mobA(Tn) gene and RK231 must be present in trans. With alkaline agarose gel electrophoresis and filter blot hybridizations, it was shown that at oriT(Tn) there was a site- and strand-specific cleavage event that was dependent on mobA(Tn). The 5' end of this cleavage site was mapped by primer extension, and the nucleotide sequence surrounding the site had homology to a family of oriT nick sites found in mobilizable plasmids of gram-positive bacteria. Removal of the nick site by deletion of 18 bp surrounding the site resulted in a significant loss of transfer activity.

Comprehensive Sequence Analyses of 5' Flanking Regions of Primate Alu Elements.

Retrotransposons have been generally known to integrate randomly into host genomes. Jurka (Proc. Natl. Acad. Sci. USA 94: 1872-1877, 1977), however, showed consensus sequence patterns at integration sites of certain mammalian retrotransposons, and suggested involvement of sequence specific enzymes that mediate integration. We have conducted comprehensive sequence analyses of 5' flanking regions of primate Alu elements. In contrast to the small but clean data set Jurka (1977) used, (1) larger number of samples were used, (2) wider region of 5' end of Alu elements was analyzed, and (3) comparisons were made among different subfamilies for comprehensive analyses in order to identify characteristic sequence pattern(s) preceding 5' end of Alu elements. The nucleotide occurrences at each position within 500 bases of 5' end of Alus were counted to obtain profiles. Information content at each nucleotide position in the same region was, then, computed. Distinctive difference in the nucleotide composition and information content values that divides the region into two was observed. The region between -20 and 5' end of Alu elements is found to be highly adenine-rich and shows significantly higher information content values compared to the rest of the region, implying the existence of certain characteristic sequence pattern in this region. Also, younger subfamilies of Alu elements show higher information content values than older subfamilies. This implies that certain characteristic sequence pattern already existed in the region between -20 and 5' end of Alu elements at the time of Alu integration, and accumulation of mutation in the course of time resulted in the less distinctive sequence pattern in older sequences. Frequencies of all possible triplets (total of 64) were measured in the same region in order to identify characteristic sequence pattern(s). Observation that frequencies of triplets aaa, taa and tta in the 5' flanking sequences were high is consistent with Jurka (1977). Frequencies of some other triplets such as gaa, caa, aac, ctt, gtt, atg, etc. which do not comprise the primary candidates for the nick site in Jurka (1977) also show significantly high frequencies.

Directed integration of minute virus of mice DNA into episomes.

Recent studies with adeno-associated virus (AAV) have shown that site-specific integration is directed by DNA sequence motifs that are present in both the viral replication origin and the chromosomal preintegration DNA and that specify binding and nicking sites for the viral regulatory Rep protein. This finding raised the question as to whether other parvovirus regulatory proteins might direct site-specific recombination with DNA targets that contain origin sequences functionally equivalent to those described for AAV. To investigate this question, active and inactive forms of the minute virus of mice (MVM) 3' replication origin, derived from a replicative-form dimer-bridge intermediate, were propagated in an Epstein-Barr virus-based shuttle vector which replicates as an episome in a cell-cycle-dependent manner in mammalian cells. Upon MVM infection of these cells, the infecting genome integrated into episomes containing the active-origin sequence reported to be efficiently nicked by the MVM regulatory protein NS1. In contrast, MVM did not integrate into episomes containing either the inactive form of the origin sequence reported to be inefficiently nicked by NS1 or the active form from which the NS1 consensus nick site had been deleted. The structure of the cloned MVM episomal recombinants displayed several features previously described for AAV episomal and chromosomal recombinants. The findings indicate that the rules which govern AAV site-specific recombination also apply to MVM and suggest that site-specific chromosomal insertions may be achievable with different autonomous parvovirus replicator proteins which recognize binding and nicking sites on the target DNA.

Mutational analysis of the R64 oriT region: requirement for precise location of the NikA-binding sequence.

Conjugative DNA transfer of IncI1 plasmid R64 is initiated by the introduction of a site- and strand-specific nick into the origin of transfer (oriT). In R64 oriT, 17-bp (repeat A and B) and 8-bp inverted-repeat sequences with mismatches are located 8 bp away from the nick site. The nicking is mediated by R64 NikA and NikB proteins. To analyze the functional organization of the R64 oriT region, various deletion, insertion, and substitution mutations were introduced into a 92-bp minimal R64 oriT sequence and their effects on oriT function were investigated. This detailed analysis confirms our previous prediction that the R64 oriT region consists of an oriT core sequence and additional sequences necessary for full oriT activity. The oriT core sequence consists of the repeat A sequence, which is recognized by R64 NikA protein, and the nick region sequence, which is conserved among various origins of transfer and is most probably recognized by NikB protein. The oriT core sequence is sufficient for NikAB-mediated oriT-specific nicking. Furthermore, it was shown that the repeat A sequence is essential for localization to a precise position relative to the nick site for oriT function. This seems to be required for the formation of a functional ternary complex consisting of NikA and NikB proteins and oriT DNA. The repeat B sequence and 8-bp inverted repeat sequences are suggested to be required for the termination of DNA transfer.

Roles of TraI protein with activities of cleaving and rejoining the single-stranded DNA in both initiation and termination of conjugal DNA transfer.

The plasmid R100 encodes the TraI protein, which is required for conjugal DNA transfer. TraI has the activity of site- and strand-specific nicking of the supercoiled plasmid DNA. The molecular mechanism of this specific nicking, which is supposed to be the initiation reaction of DNA transfer, is not understood. We have demonstrated that TraI has the ability to cleave the single-stranded DNA at the same site as the nicking site (nic) in a region, which we here refer to as sbi. The product contained the TraI protein which was covalently linked to the newly generated 5' end of the nicking reaction. Both the cleaving and nicking reactions took place under almost the same conditions and required the presence of the sbi region. DNase I-footprinting analysis revealed that the TraI bound to the single-stranded DNA of the sbi region. TraI did not cleave the double-stranded DNA fragment, but it did cleave the double-stranded DNA with a single-stranded DNA portion in the sbi region. KMnO4 mapping analysis revealed that TraI can melt the sbi region in the supercoiled DNA to generate a single-stranded portion. We have also demonstrated that TraI was able to rejoin the cleaved products. The rejoining reaction required the 5' end of one cleaved product with the TraI covalently attached and the 3' end of the other product containing the sbi region. Our results demonstrate that the nicking reaction-the initiation reaction of DNA transfer-is actually the cleaving reaction of the single-stranded DNA. TraI, which has both cleaving and rejoining activities, is thought to be involved in the termination of DNA transfer, to give a copy of the conjugative plasmid by joining the 5' end, which is generated by the initiation reaction, with the 3' end, which will be generated upon cleavage of the sbi region appearing after one round of the rolling circle replication of the plasmid.

Susceptibility of Human Sperm toin SituDNA Denaturation Is Strongly Correlated with DNA Strand Breaks Identified by Single-Cell Electrophoresis

Susceptibility of mammalian sperm DNA to low pH- or heat-induced denaturationin situhas shown very strong dose–response relationships with animal and human exposure to chemical and physical toxicants and also fertility potential. In this study, 23 human semen samples representing a wide range in percentage (7–86%) of sperm exhibiting abnormally high susceptibility of DNAin situto denaturation were studied for the integrity of their DNA using alkaline comet assay (single-cell microgel electrophoresis, pH 10.0). The percentage of comets observed for these samples ranged from 5 to 95%; these data correlated strongly with the percentage of sperm with increased DNA denaturability (r= 0.973;P< 0.001). Labeling of 3′ ends of nicked DNA sites with 5-bromo-2′-deoxyuridine 5′-triphosphate (BrdUTP) followed by tagging with FITC–BrdUTP monoclonal antibody and flow cytometry also indicated significantly strong correlations of BrdUTP incorporation with both abnormal susceptibility of DNA to denaturation (r= 0.859,P< 0.001) and comet assay (r= 0.812,P< 0.001). The relationship among susceptibility of sperm chromatin to acid denaturationin situ,BrdUTP incorporation, and formation of comets suggests that DNA fragmentation monitored by these assays may have important physiological relevance in terms of sperm quality and fertility potential.

Blocking rolling circle replication with a UV lesion creates a deletion hotspot.

UV light irradiation increases genetic instability by causing mutations and deletions. The mechanism of UV-induced rearrangements was investigated making use of deletion-prone plasmids. Chimeric plasmids carrying pBR322 and M13 replication origins undergo deletions that join the M13 replication origin to a random nucleotide. A restriction fragment was UV irradiated, introduced into such a hybrid plasmid and deletions formed at the M13 origin were analysed. In most of the deletant molecules, the M13 replication nick site was linked to a nucleotide in the irradiated fragment, showing that UV lesions are deletion hotspots. These deletions were independent of the UvrABC excision repair proteins, suggesting that the deletogenic structure is the lesion itself and not a repair intermediate. They were not found in the absence of M13 replication, indicating that they result from the encounter of the M13 replication fork with the UV lesion. Furthermore, UV-induced deletions occurred independently of pBR322 replication. We conclude that, in contrast to pBR322 replication forks, M13 replication forks blocked by UV lesions are deletion prone. We propose that the deletion-prone properties of a UV-arrested polymerase depend on the associated helicase.

Genetic Evidence That Recognition of cosQ the Signal for Termination of Phage λ DNA Packaging, Depends on the Extent of Head Filling

Packaging a phage lambda chromosome involves cutting the chromosome from a concatemer and translocating the DNA into a prohead. The cutting site, cos, consists of three subsites: cosN, the nicking site; cosB, a site required for packaging initiation; and cosQ a site required for termination of packaging. cosB contains three binding sites (R sequences) for gpNu1, the small subunit of terminase. Because cosQ has sequence identity to the R sequences, it has been proposed that cosQ is also recognized by gpNu1. Suppressors of cosB mutations were unable to suppress a cosQ point mutation. Suppressors of a cosQ mutation (cosQ1) were isolated and found to be of three sorts, the first affecting a base pair in cosQ. The second type of cosQ suppression involved increasing the length of the phage chromosome to a length near to the maximum capacity of the head shell. A third class of suppressors were missense mutations in gene B, which encodes the portal protein of the virion. It is speculated that increasing DNA length and altering the portal protein may reduce the rate of translocation, thereby increasing the efficiency of recognition of the mutant cosQ. None of the cosQ suppressors was able to suppress cosB mutations. Because cosQ and cosB mutations are suppressed by very different types of suppressors, it is concluded that cosQ and the R sequences of cosB are recognized by different DNA-binding determinants.

Functional features of an ssi signal of plasmid pGKV21 in Escherichia coli.

A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13 delta lac182) lacking the complementary-strand origin in Escherichia coli. This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin. SSI activity is orientation specific with respect to the direction of replication. We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that E. coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E. coli single-stranded DNA binding (SSB) protein. This region contains two stem-loop structures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E. coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E. coli RNA polymerase on the SSB-coated single-stranded DNA template.

The relaxosome protein MobC promotes conjugal plasmid mobilization by extending DNA strand separation to the nick site at the origin of transfer.

The frequency of conjugal mobilization of plasmid R1162 is decreased approximately 50-fold if donor cells lack MobC, one of the plasmid-encoded proteins making up the relaxosome at the origin of transfer (oriT). The absence of MobC has several different effects on oriT DNA. Site- and strand-specific nicking by MobA protein is severely reduced, accounting for the lower frequency of mobilization. The localized DNA strand separation required for this nicking is less affected, but becomes more sensitive to the level of active DNA gyrase in the cell. In addition, strand separation is not efficiently extended through the region containing the nick site. These effects suggest a model in which MobC acts as a molecular wedge for the relaxosome-induced melting of oriT DNA. The effect of MobC on strand separation may be partially complemented by the helical distortion induced by supercoiling. However, MobC extends the melted region through the nick site, thus providing the single-stranded substrate required for cleavage by MobA.

Why is the initiation nick site of an AT-rich rolling circle plasmid at the tip of a GC-rich cruciform?

pT181 and other closely related rolling circle plasmids have the nicking site for initiation of replication between the arms of a GC-rich inverted repeat sequence adjacent to the binding site for the dimeric initiator protein. Replication is initiated by the initiator-induced extrusion of this sequence as a cruciform, creating a single-stranded region for nicking by the protein. Nicking is followed by assembly of the replisome without relaxation of the secondary structure. Following termination, the initiator protein is released with a short oligonucleotide attached to one subunit, which prevents it from being recycled, a necessary feature of the plasmid's replication control system. The modified initiator can cleave single-stranded substrates and can nick and relax supercoiled plasmid DNA weakly. Although it can bind to its recognition sequence in the leading strand origin, the modified protein cannot induce cruciform extrusion, and it is proposed that this inability is the key to understanding the biological rationale for having the nicking site at the tip of a cruciform: the need to provide the functional initiator with a catalytic advantage over the modified one sufficient to offset the numerical advantage and metabolic stability of the latter.

Mass spectrometric characterization of nicked fragments of the β-subunit of human chorionic gonadotropin

We describe the use of an HPLC/MS technique for the characterization of nicked fragments of hCG beta-subunit. After reductive alkylation of the nicked hCG beta-subunit with vinylpyridine, endoproteinase Glu-C or trypsin was used to digest the protein to produce peptides that could be analyzed by HPLC/electrospray ionization MS. Human leukocyte elastase digestion was used to produce an experimentally nicked hCG. Two nicking sites were observed, between amino acids 42Thr and 43Arg and between 44Val and 45Leu. The former site has not been previously reported for elastase digestion. The structures of the fragments were confirmed by HPLC/MS after removal of the oligosaccharide by direct mass measurement and by mass determination of their proteolytic digests. Without the glycopeptidase treatment, the microheterogeneity of the two N-linked oligosaccharides could be deduced from the spectra of the proteolytic fragments. Nicking with elastase was found to alter the oligosaccharide structures. Nicked beta-subunit samples isolated from the urine of choriocarcinoma patients were also analyzed and the location of the nicking site(s) agreed with that determined by classical techniques. Important differences in the oligosaccharide structures were also observed in these samples, including the presence of triantennary oligosaccharides not found in hCG from healthy subjects. These findings demonstrate the potential of HPLC/MS for characterization of glycoprotein standard preparations.

Nucleotide sequence of a nicking site of the Streptomyces plasmid pSN22 replicating by the rolling circle mechanism

A putative nicking site in the double strand origin (DSO) of the Streptomyces plasmid pSN22 was identified by comparing the nucleotide sequence of the DSO region with those of two other Streptomyces plasmids, pIJ101 and pJV1. A 7-bp sequence of this putative nicking site, 5′-CTTGGGA-3′, was similar to the consensus sequence of the nicking site of the pC194 group of plasmids. When several point mutations were introduced into this 7-bp sequence, the transformation abilities of the mutant plasmid molecules for Streptomyces lividans were either reduced or lost. Southern hybridization analysis indicated that these mutant plasmids could not replicate in S. lividans, but were integrated into the chromosomal DNA.

Sequence requirements for the termination of rolling-circle replication of plasmid pT181.

Most small multicopy plasmids of Gram-positive bacteria and many in Gram-negative bacteria replicate by a rolling-circle (RC) mechanism. The replication initiator proteins encoded by the RC plasmids and single-stranded bacteriophages of Escherichia coli have origin-specific nicking-closing activities that are required for the initiation and termination of RC replication. We have investigated the sequence requirements for termination of RC replication of plasmid pT181. The initiator nick site is located in the loop of a hairpin region (IRII) within the pT181 origin of replication. By mutational analysis, we have found that several nucleotides within the stem of IRII which are critical for the initiation activity are dispensable for termination of replication. We also demonstrate that nucleotides in the right arm of IRII, but not the left arm, are absolutely required for termination of RC replication. We have also identified specific nucleotides in IRII that are critical for its termination activity. The sequence of the right arm of the hairpin must be located downstream of the initiator nick site for termination, suggesting that termination requires a specific orientation of the initiator protein at the origin.

Sequence patterns indicate an enzymatic involvement in integration of mammalian retroposons.

It is commonly accepted that the reverse-transcribed cellular RNA molecules, called retroposons, integrate at staggered breaks in mammalian chromosomes. However, unlike what was previously thought, most of the staggered breaks are not generated by random nicking. One of the two nicks involved is primarily associated with the 5'-TTAAAA hexanucleotide and its variants derived by a single base substitution, particularly A --> G and T --> C. It is probably generated in the antisense strand between the consensus bases 3'-AA and TTTT complementary to 5'-TTAAAA. The sense strand is nicked at variable distances from the TTAAAA consensus site toward the 3' end, preferably within 15-16 base pairs. The base composition near the second nicking site is also nonrandom at positions preceding the nick. On the basis of the observed sequence patterns it is proposed that integration of mammalian retroposons is mediated by an enzyme with endonucleolytic activity. The best candidate for such enzyme may be the reverse transcriptase encoded by the L1 non-long-terminal-repeat retrotransposon, which contains a freshly reported domain homologous to the apurinic/apyrimidinic (AP) endonuclease family [Martin, F., Olivares, M., Lopez, M. C. & Alonso, C. (1996) Trends Biochem. Sci. 21, 283-285; Feng, Q., Moran, J. V., Kazazian, H. H. & Boeke, J. D. (1996) Cell 87, 905-916] and shows nicking in vitro with preference for targets similar to 5'-TTAAAA/3'-AATTTT consensus sequence [Feng, Q., Moran, J. V., Kazazian, H. H. & Boeke, J. D. (1996) Cell 87, 905-916]. A model for integration of mammalian retroposons based on the presented data is discussed.

The mobilization protein, MobM, of the streptococcal plasmid pMV158 specifically cleaves supercoiled DNA at the plasmid oriT

The streptococcal plasmid pMV158 replicates by the rolling circle mechanism. It encodes a relaxase protein of 494 residues, termed MobM, involved in conjugative mobilization. MobM protein was overproduced, purified, and shown specifically to relax supercoiled pMV158 DNA. The 5′-end and the 3′-end of the nick site introduced by MobM have been determined by sequencing and by primer extension analysis. The nucleophilic attack exerted by MobM is in the 5′-GpT-3′ dinucleotide, within the sequence 5′-TAGTGTG/TTA-3′. Upon cleavage, MobM protein remains tightly associated with its target DNA, probably through a covalent bond. The pMV158 oriT did not exhibit homologies with known origins of transfer of plasmids from Gram-negative bacteria. However, several plasmids from Gram-positive hosts have a region identical or very similar to the pMV158 oriT. To our knowledge, this is the first demonstration of a relaxase activity of a mobilization protein from a plasmid replicating by the rolling circle mechanism.

Relationship between a low toxicity of the mutant A subunit of enterotoxigenic Escherichia coli enterotoxin and its strong adjuvant action.

In the work described here it was determined if and how unnicking in the A subunit of Escherichia coli enterotoxin at Arg192 or nearby residues affected biological activities of the toxin. The mutant toxin was constructed to lack the nick site in the A subunit by deleting the tripeptide Arg192-Thr193-Ile194, which is essential for toxicity. The mutant toxin did not exhibit agmatine ADP-ribosyltransferase activity in the presence or absence of the ADP-ribosylation factor and had less diarrhoeal activity and lower induction of cyclic AMP than did LT. The mutant toxin exhibited a much stronger adjuvant action on antibody responses to measles virus, keyhole limpt haemocyanin, bovine immunoglobulin and ovalbumin compared with LT. The altered toxicity of the mutant toxin might be closely related to the potent adjuvant action on antibody responses to antigens. The relationship between two activities is discussed.

Unique features of the mitochondrial rolling circle-plasmid mp1 from the higher plant Chenopodium album (L.).

We analyzed the structure and replication of the mitochondrial (mt) circular DNA plasmid mp1 (1309 bp) from the higher plant Chenopodium album(L.). Two dimensional gel electrophoresis (2DE) revealed the existence of oligomers of up to a decamer in addition to the prevailing monomeric form. The migration behavior of cut replication intermediates during 2DE was consistent with a rolling circle (RC) type of replication. We detected entirely single-stranded (ss) plasmid copies hybridizing only with one of the two DNA strands. This result indicates the occurence of an asymmetric RC replication mechanism. mp1 has, with respect to its replication, some unique features compared with bacterial RC plasmids. We identified and localized a strand-specific nicking site (origin of RC replication) on the plasmid by primer extension studies. Nicks in the plasmid were found to occur at any one of six nucleotides (TAAG/GG) around position 735 of the leading strand. This sequence shows no homology to origin motifs from known bacterial RC replicons. mp1 is the first described RC plasmid in a higher plant.