Pathways and signatures of mutagenesis at targeted DNA nicks

Nicks are the most frequent form of DNA damage and a potential source of mutagenesis in human cells. By deep sequencing, we have identified factors and pathways that promote and limit mutagenic repair at a targeted nick in human cells. Mutations were distributed asymmetrically around the nick site. BRCA2 inhibited all categories of mutational events, including indels, SNVs and HDR. DNA2 and RPA promoted resection. DNA2 inhibited 1 bp deletions but contributed to longer deletions, as did REV7. POLQ stimulated SNVs. Parallel analysis of DSBs targeted to the same site identified similar roles for DNA2 and POLQ (but not REV7) in promoting deletions and for POLQ in stimulating SNVs. Insertions were infrequent at nicks, and most were 1 bp in length, as at DSBs. The translesion polymerase REV1 stimulated +1 insertions at one nick site but not another, illustrating the potential importance of sequence context in determining the outcome of mutagenic repair. These results highlight the potential for nicks to promote mutagenesis, especially in BRCA-deficient cells, and identify mutagenic signatures of DNA2, REV1, REV3, REV7 and POLQ.

Impact of substrate-template stability, temperature, phosphate location, and nick-site base pairs on non-enzymatic DNA ligation: Defining parameters for optimization of ligation rates and yields with carbodiimide activation

ABSTRACTNon-enzymatic, chemical ligation is an important tool for the generation of synthetic DNA structures, which are used for a wide range of applications. Surprisingly, reported chemical ligation yields range from 30% to 95% for the same chemical activating agent and comparable DNA structures. We report a systematic study of DNA ligation using a well-defined bimolecular test system and water-soluble carbodiimide (EDC) as a phosphate-activating agent. Our results reveal interplay between template-substrate stability and the rates of the chemical steps of ligation, which can cause yields to increase or decrease with increasing temperature. Phosphate location at the nick site also exhibits a strong influence on ligation rates and yields, with a 3’ phosphate providing yields near 100% after 24 hours for particularly favourable reaction conditions, while comparable reactions with the phosphate on the 5’ position of the nick site only reach 40% ligation even after 48 hours. Ligation rates are also shown to be sensitive to the identity of base pairs flanking a nick site, with some varying by more than three-fold. Finally, DNA substrate modification by EDC can, in some cases, make long reaction times and repeated addition of EDC an ineffective strategy for increasing ligation yields.

Genetic and Functional Analyses of the mob Operon on Conjugative Transposon CTn341 from Bacteroides spp.

ABSTRACT Bacteroides are Gram-negative anaerobes indigenous to the intestinal tract of humans, and they are important opportunistic pathogens. Mobile genetic elements, such as conjugative transposons (CTns), have contributed to an increase in antibiotic resistance in these organisms. CTns are self-transmissible elements that belong to the superfamily of integrative and conjugative elements (ICEs). CTn341 is 52 kb; it encodes tetracycline resistance and its transfer is induced by tetracycline. The mobilization region of CTn341 was shown to be comprised of a three-gene operon, mobABC, and the transfer origin, oriT. The three genes code for a nicking accessory protein, a relaxase, and a VirD4-like coupling protein, respectively. The Mob proteins were predicted to mediate the formation of the relaxosome complex, nick DNA at the oriT, and shuttle the DNA/protein complex to the mating-pore apparatus. The results of mutational studies indicated that the three genes are required for maximal transfer of CTn341. Mob gene transcription was induced by tetracycline, and this regulation was mediated through the two-component regulatory system, RteAB. The oriT region of CTn341 was located within 100 bp of mobA, and a putative Bacteroides consensus nicking site was observed within this region. Mutation of the putative nick site resulted in a loss of transfer. This study demonstrated a role of the mobilization region for transfer of Bacteroides CTns and that tetracycline induction occurs for the mob gene operon, as for the tra gene operon(s), as shown previously.

Analysis of the Mobilization Functions of the Vancomycin Resistance Transposon Tn1549, a Member of a New Family of Conjugative Elements

ABSTRACT Conjugative transfer from Clostridium symbiosum to enterococci of Tn1549, which confers VanB-type vancomycin resistance, has been reported. This indicates the presence of a transfer origin (oriT) in the element. Transcription analysis of Tn1549 indicated that orf29, orf28, orfz, and orf27 were cotranscribed. A pACYC184 derivative containing 250 bp intergenic to orf29-orf30 of Tn1549 was mobilized in Escherichia coli recA::RP4::Δnic provided that orf28 and orf29 were delivered simultaneously. These open reading frame (ORF) genes were able to promote mobilization in trans, but a cis-acting preference was observed. On the basis of a mobilization assay, a minimal 28-bp oriT was delimited, although the frequency of transfer was significantly reduced compared to that of a 130-bp oriT fragment. The minimal oriT contained an inverted repeat and a core, which was homologous to the cleavage sequence found in certain Gram-positive rolling-circle replicating (RCR) plasmids. While Orf29 was a mobilization accessory component similar to MobC proteins, Orf28 was identified as a relaxase belonging to a new phyletic cluster of the MOBp superfamily. The nick site was identified within oriT by an oligonucleotide cleavage assay. Closely related oriTs linked to mobilization genes were detected in data banks; they were found in various integrative and conjugative elements (ICEs) originating mainly from anaerobes. These results support the notion that Tn1549 is a member of a MOBp clade. Interestingly, the Tn1549-derived constructs were mobilized by RP4 in E. coli, suggesting that a relaxosome resulting from DNA cleavage by Orf28 interacted with the coupling protein TraG. This demonstrates the capacity of Tn1549 to be mobilized by a heterologous transfer system.

Precise Determination, Cross-Recognition, and Functional Analysis of the Double-Strand Origins of the Rolling-Circle Replication Plasmids in Haloarchaea

ABSTRACT The precise nick site in the double-strand origin (DSO) of pZMX201, a 1,668-bp rolling-circle replication (RCR) plasmid from the haloarchaeon Natrinema sp. CX2021, was determined by electron microscopy and DSO mapping. In this plasmid, DSO nicking occurred between residues C404 and G405 within a heptanucleotide sequence (TCTC/GGC) located in the stem region of an imperfect hairpin structure. This nick site sequence was conserved among the haloarchaeal RCR plasmids, including pNB101, suggesting that the DSO nick site might be the same for all members of this plasmid family. Interestingly, the DSOs of pZMX201 and pNB101 were found to be cross-recognized in RCR initiation and termination in a hybrid plasmid system. Mutation analysis of the DSO from pZMX201 (DSOZ) in this hybrid plasmid system revealed that: (i) the nucleotides in the middle of the conserved TCTCGGC sequence play more-important roles in the initiation and termination process; (ii) the left half of the hairpin structure is required for initiation but not for termination; and (iii) a 36-bp sequence containing TCTCGGC and the downstream sequence is essential and sufficient for termination. In conclusion, these haloarchaeal plasmids, with novel features that are different from the characteristics of both single-stranded DNA phages and bacterial RCR plasmids, might serve as a good model for studying the evolution of RCR replicons.

The Relaxase of the Rhizobium etli Symbiotic Plasmid Shows nic Site cis-Acting Preference

ABSTRACT Genetic and biochemical characterization of TraA, the relaxase of symbiotic plasmid pRetCFN42d from Rhizobium etli, is described. After purifying the relaxase domain (N265TraA), we demonstrated nic binding and cleavage activity in vitro and thus characterized for the first time the nick site (nic) of a plasmid in the family Rhizobiaceae. We studied the range of N265TraA relaxase specificity in vitro by testing different oligonucleotides in binding and nicking assays. In addition, the ability of pRetCFN42d to mobilize different Rhizobiaceae plasmid origins of transfer (oriT) was examined. Data obtained with these approaches allowed us to establish functional and phylogenetic relationships between different plasmids of this family. Our results suggest novel characteristics of the R. etli pSym relaxase for previously described conjugative systems, with emphasis on the oriT cis-acting preference of this enzyme and its possible biological relevance.

TraA and its N-terminal relaxase domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as dimers in solution

TraA is the DNA relaxase encoded by the broad-host-range Grampositive plasmid pIP501. It is the second relaxase to be characterized from plasmids originating from Gram-positive organisms. Full-length TraA (654 amino acids) and the N-terminal domain (246 amino acids), termed TraAN246, were expressed as 6×His-tagged fusions and purified. Small-angle X-ray scattering and chemical cross-linking proved that TraAN246 and TraA form dimers in solution. Both proteins revealed oriTpIP501 (origin of transfer of pIP501) cleavage activity on supercoiled plasmid DNA in vitro. oriT binding was demonstrated by electrophoretic mobility shift assays. Radiolabelled oligonucleotides covering different parts of oriTpIP501 were subjected to binding with TraA and TraAN246. The KD of the protein–DNA complex encompassing the inverted repeat, the nick site and an additional 7 bases was found to be 55 nM for TraA and 26 nM for TraAN246. The unfolding of both protein constructs was monitored by measuring the change in the CD signal at 220 nm upon temperature change. The unfolding transition of both proteins occurred at approx. 42 °C. CD spectra measured at 20 °C showed 30% α-helix and 13% β-sheet for TraA, and 27% α-helix and 18% β-sheet content for the truncated protein. Upon DNA binding, an enhanced secondary structure content and increased thermal stability were observed for the TraAN246 protein, suggesting an induced-fit mechanism for the formation of the specific relaxase–oriT complex.

Genome Packaging Sense Is Controlled by the Efficiency of the Nick Site in the Right-End Replication Origin of Parvoviruses Minute Virus of Mice and LuIII

ABSTRACT The parvovirus minute virus of mice (MVM) packages predominantly negative-sense single strands, while its close relative LuIII encapsidates strands of both polarities with equal efficiency. Using genomic chimeras and mutagenesis, we show that the ability to package positive strands maps not, as originally postulated, to divergent untranslated regions downstream of the capsid gene but to the viral hairpins and predominantly to the nick site of OriR, the right-end replication origin. In MVM, the sequence of this site is 5′-CTAT▾TCA-3′, while in LuIII a two-base insertion (underlined) changes it to 5′-CTAT AT ▾TCA-3′. Matched LuIII genomes differing only at this position (designated LuIII and LuΔ2) packaged 47 and <8% positive-sense strands, respectively. OriR sequences from these viruses were both able to support NS1-mediated nicking in vitro, but initiation efficiency was consistently two- to threefold higher for LuΔ2 derivatives, suggesting that LuIII's ability to package positive strands is determined by a suboptimal right-end origin rather than by strand-specific packaging sequences. These observations support a mathematical “kinetic hairpin transfer” model, previously described by Chen and colleagues (K. C. Chen, J. J. Tyson, M. Lederman, E. R. Stout, and R. C. Bates, J. Mol. Biol. 208:283-296, 1989), that postulates that preferential excision of particular strands is solely responsible for packaging specificity. By analyzing replicative-form (RF) DNA generated in vivo during LuIII and LuΔ2 infections, we extend this model, showing that positive-sense strands do accumulate in LuΔ2 infections as part of duplex RF DNA, but these do not support packaging. However, replication is biphasic, so that accumulation of positive-sense strands is ultimately suppressed, probably because the onset of packaging removes newly displaced single strands from the replicating pool.