Effects of Adeno-Associated Virus DNA Hairpin Structure on Recombination

ABSTRACT Hairpin DNA ends are evolutionarily conserved intermediates in DNA recombination. The hairpin structures present on the ends of the adeno-associated virus (AAV) genome are substrates for recombination that give rise to persistent circular and concatemeric DNA episomes through intramolecular and intermolecular recombination, respectively. We have developed circularization-dependent and orientation-specific self-complementary AAV (scAAV) vectors as a reporter system to examine recombination events involving distinct hairpin structures, i.e., closed versus open hairpins. The results suggest that intramolecular recombination (circularization) is far more efficient than intermolecular recombination (concatemerization). Among all possible combinations of terminal repeats (TRs) involved in intermolecular recombination, the closed-closed TR structures are twice as efficient as the open-open TR substrates for recombination. In addition, both intramolecular recombination and intermolecular recombination exhibit the common dependency on specific DNA polymerases and topoisomerases. The circularization-dependent and orientation-specific scAAV vectors can serve as an efficient and controlled system for the delivery of DNA structures that mimic mammalian recombination intermediates and should be useful in assaying recombination in different experimental settings as well as elucidating the molecular mechanism of recombinant AAV genome persistence.

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Host Cell DNA Repair Pathways in Adeno-Associated Viral Genome Processing

Journal of Virology ◽

10.1128/jvi.00841-06 ◽

2006 ◽

Vol 80 (21) ◽

pp. 10346-10356 ◽

Cited By ~ 68

Author(s):

Vivian W. Choi ◽

Douglas M. McCarty ◽

R. Jude Samulski

Keyword(s):

Dna Repair ◽

Dna Recombination ◽

Recq Helicase ◽

Cellular Mechanisms ◽

Adeno Associated Virus ◽

Mrn Complex ◽

Dependent Vector ◽

Cellular Dna ◽

Dna Maintenance

ABSTRACT Recentstudies have shown that wild-type and recombinant adeno-associated virus (AAV and rAAV) genomes persist in human tissue predominantly as double-stranded (ds) circular episomes derived from input linear single-stranded virion DNA. Using self-complementary recombinant AAV (scAAV) vectors, we generated intermediates that directly transition to ds circular episomes. The scAAV genome ends are palindromic hairpin-structured terminal repeats, resembling a double-stranded break repair intermediate. Utilizing this substrate, we found cellular DNA recombination and repair factors to be essential for generating circular episomal products. To identify the specific cellular proteins involved, the scAAV circularization-dependent vector was used as a reporter in 19 mammalian DNA repair-deficient cell lines. The results show that RecQ helicase family members (BLM and WRN), Mre11 and NBS1 of the Mre11-Rad50-Nbs1 (MRN) complex, and ATM are required for efficient scAAV genome circularization. We further demonstrated that the scAAV genome requires ATM and DNA-PKCS, but not NBS1, to efficiently convert to a circular form in nondividing cells in vivo using transgenic mice. These studies identify specific pathways involved for further elucidating viral and cellular mechanisms of DNA maintenance important to the viral life cycle and vector utilizations.

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Determination of Anti-Adeno-Associated Virus Vector Neutralizing Antibody Titer with an In Vitro Reporter System

Human Gene Therapy Methods ◽

10.1089/hgtb.2015.037 ◽

2015 ◽

Vol 26 (2) ◽

pp. 45-53 ◽

Cited By ~ 40

Author(s):

Amine Meliani ◽

Christian Leborgne ◽

Sabrina Triffault ◽

Laurence Jeanson-Leh ◽

Philippe Veron ◽

...

Keyword(s):

Antibody Titer ◽

Neutralizing Antibody ◽

Virus Vector ◽

Reporter System ◽

Adeno Associated Virus

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Znc2 module of RAG1 contributes towards structure-specific nuclease activity of RAGs

Biochemical Journal ◽

10.1042/bcj20200361 ◽

2020 ◽

Vol 477 (18) ◽

pp. 3567-3582

Author(s):

Namrata M. Nilavar ◽

Mayilaadumveettil Nishana ◽

Amita M. Paranjape ◽

Raghunandan Mahadeva ◽

Rupa Kumari ◽

...

Keyword(s):

Signal Sequence ◽

Specific Binding ◽

Dna Recombination ◽

Point Mutations ◽

Nuclease Activity ◽

Heteroduplex Dna ◽

Central Domain ◽

Dna Structures ◽

Recombination Activating Genes ◽

Catalytic Motif

Recombination activating genes (RAGs), consisting of RAG1 and RAG2 have ability to perform spatially and temporally regulated DNA recombination in a sequence specific manner. Besides, RAGs also cleave at non-B DNA structures and are thought to contribute towards genomic rearrangements and cancer. The nonamer binding domain of RAG1 binds to the nonamer sequence of the signal sequence during V(D)J recombination. However, deletion of NBD did not affect RAG cleavage on non-B DNA structures. In the present study, we investigated the involvement of other RAG domains when RAGs act as a structure-specific nuclease. Studies using purified central domain (CD) and C-terminal domain (CTD) of the RAG1 showed that CD of RAG1 exhibited high affinity and specific binding to heteroduplex DNA, which was irrespective of the sequence of single-stranded DNA, unlike CTD which showed minimal binding. Furthermore, we show that ZnC2 of RAG1 is crucial for its binding to DNA structures as deletion and point mutations abrogated the binding of CD to heteroduplex DNA. Our results also provide evidence that unlike RAG cleavage on RSS, central domain of RAG1 is sufficient to cleave heteroduplex DNA harbouring pyrimidines, but not purines. Finally, we show that a point mutation in the DDE catalytic motif is sufficient to block the cleavage of CD on heteroduplex DNA. Therefore, in the present study we demonstrate that the while ZnC2 module in central domain of RAG1 is required for binding to non-B DNA structures, active site amino acids are important for RAGs to function as a structure-specific nuclease.

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Damage Tolerance Protein Mus81 Associates with the FHA1 Domain of Checkpoint Kinase Cds1

Molecular and Cellular Biology ◽

10.1128/mcb.20.23.8758-8766.2000 ◽

2000 ◽

Vol 20 (23) ◽

pp. 8758-8766 ◽

Cited By ~ 198

Author(s):

Michael N. Boddy ◽

Antonia Lopez-Girona ◽

Paul Shanahan ◽

Heidrun Interthal ◽

Wolf-Dietrich Heyer ◽

...

Keyword(s):

Damage Tolerance ◽

Excision Repair ◽

Protein Docking ◽

Serine Threonine Kinase ◽

Dna Structures ◽

Threonine Kinase ◽

Repair Protein ◽

Nucleotide Excision ◽

Evolutionarily Conserved ◽

Genetic Epistasis

ABSTRACT Cds1, a serine/threonine kinase, enforces the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. Cds1 is required for survival of replicational stress caused by agents that stall replication forks, but how Cds1 performs these functions is largely unknown. Here we report that the forkhead-associated-1 (FHA1) protein-docking domain of Cds1 interacts with Mus81, an evolutionarily conserved damage tolerance protein. Mus81 has an endonuclease homology domain found in the XPF nucleotide excision repair protein. Inactivation of mus81 reveals a unique spectrum of phenotypes. Mus81 enables survival of deoxynucleotide triphosphate starvation, UV radiation, and DNA polymerase impairment. Mus81 is essential in the absence of Bloom's syndrome Rqh1 helicase and is required for productive meiosis. Genetic epistasis studies suggest that Mus81 works with recombination enzymes to properly replicate damaged DNA. Inactivation of Mus81 triggers a checkpoint-dependent delay of mitosis. We propose that Mus81 is involved in the recruitment of Cds1 to aberrant DNA structures where Cds1 modulates the activity of damage tolerance enzymes.

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An Alternative Method for Evaluating Stabilities of DNA Hairpin Structures

Bulletin of the Chemical Society of Japan ◽

10.1246/bcsj.20150120 ◽

2015 ◽

Vol 88 (9) ◽

pp. 1314-1316 ◽

Cited By ~ 2

Author(s):

Hao Zhang ◽

Sai Ba ◽

Sarajane Co Co ◽

Jasmine Yiqin Lee ◽

Juanjuan Guo ◽

...

Keyword(s):

Dna Hairpin ◽

Alternative Method ◽

Hairpin Structures

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Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615439114 ◽

2017 ◽

Vol 114 (4) ◽

pp. E466-E475 ◽

Cited By ~ 22

Author(s):

Gábor M. Harami ◽

Yeonee Seol ◽

Junghoon In ◽

Veronika Ferencziová ◽

Máté Martina ◽

...

Keyword(s):

Single Molecule ◽

Domain Architecture ◽

Dna Recombination ◽

Recq Helicase ◽

Dna Structures ◽

Recq Helicases ◽

Differential Recognition ◽

D Loop ◽

Mechanistic Basis

Cells must continuously repair inevitable DNA damage while avoiding the deleterious consequences of imprecise repair. Distinction between legitimate and illegitimate repair processes is thought to be achieved in part through differential recognition and processing of specific noncanonical DNA structures, although the mechanistic basis of discrimination remains poorly defined. Here, we show thatEscherichia coliRecQ, a central DNA recombination and repair enzyme, exhibits differential processing of DNA substrates based on their geometry and structure. Through single-molecule and ensemble biophysical experiments, we elucidate how the conserved domain architecture of RecQ supports geometry-dependent shuttling and directed processing of recombination-intermediate [displacement loop (D-loop)] substrates. Our study shows that these activities together suppress illegitimate recombination in vivo, whereas unregulated duplex unwinding is detrimental for recombination precision. Based on these results, we propose a mechanism through which RecQ helicases achieve recombination precision and efficiency.

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Structure of the catalytic domain of Mre11 fromChaetomium thermophilum

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x15007566 ◽

2015 ◽

Vol 71 (6) ◽

pp. 752-757 ◽

Cited By ~ 11

Author(s):

Florian Ulrich Seifert ◽

Katja Lammens ◽

Karl-Peter Hopfner

Keyword(s):

Crystal Structure ◽

Protein Complex ◽

Catalytic Domain ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Dimer Interface ◽

Chaetomium Thermophilum ◽

Evolutionarily Conserved ◽

Dna Ends

Together with the Rad50 ATPase, the Mre11 nuclease forms an evolutionarily conserved protein complex that plays a central role in the repair of DNA double-strand breaks (DSBs). Mre11–Rad50 detects and processes DNA ends, and has functions in the tethering as well as the signalling of DSBs. The Mre11 dimer can bind one or two DNA ends or hairpins, and processes DNA endonucleolytically as well as exonucleolytically in the 3′-to-5′ direction. Here, the crystal structure of the Mre11 catalytic domain dimer fromChaetomium thermophilum(CtMre11CD) is reported. CtMre11CDcrystals diffracted to 2.8 Å resolution and revealed previously undefined features within the dimer interface, in particular fully ordered eukaryote-specific insertion loops that considerably expand the dimer interface. Furthermore, comparison with other eukaryotic Mre11 structures reveals differences in the conformations of the dimer and the capping domain. In summary, the results reported here provide new insights into the architecture of the eukaryotic Mre11 dimer.

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Failure of Hairpin-Ended and Nicked DNA To Activate DNA-Dependent Protein Kinase: Implications for V(D)J Recombination

Molecular and Cellular Biology ◽

10.1128/mcb.18.11.6853 ◽

1998 ◽

Vol 18 (11) ◽

pp. 6853-6858 ◽

Cited By ~ 25

Author(s):

Vaughn Smider ◽

W. Kimryn Rathmell ◽

Greg Brown ◽

Susanna Lewis ◽

Gilbert Chu

Keyword(s):

Protein Kinase ◽

Catalytic Subunit ◽

Cleavage Reaction ◽

Dependent Protein Kinase ◽

Dna Structures ◽

Dependent Protein ◽

Efficient Processing ◽

Dna Ends ◽

Dna Nicks

ABSTRACT V(D)J recombination is initiated by a coordinated cleavage reaction that nicks DNA at two sites and then forms a hairpin coding end and blunt signal end at each site. Following cleavage, the DNA ends are joined by a process that is incompletely understood but nevertheless depends on DNA-dependent protein kinase (DNA-PK), which consists of Ku and a 460-kDa catalytic subunit (DNA-PKCS or p460). Ku directs DNA-PKCS to DNA ends to efficiently activate the kinase. In vivo, the mouse SCID mutation in DNA-PKCSdisrupts joining of the hairpin coding ends but spares joining of the open signal ends. To better understand the mechanism of V(D)J recombination, we measured the activation of DNA-PK by the three DNA structures formed during the cleavage reaction: open ends, DNA nicks, and hairpin ends. Although open DNA ends strongly activated DNA-PK, nicked DNA substrates and hairpin-ended DNA did not. Therefore, even though efficient processing of hairpin coding ends requires DNA-PKCS, this may occur by activation of the kinase bound to the cogenerated open signal end rather than to the hairpin end itself.

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Characterization of Wild-Type Adeno-Associated Virus Type 2-Like Particles Generated during Recombinant Viral Vector Production and Strategies for Their Elimination

Journal of Virology ◽

10.1128/jvi.72.7.5472-5480.1998 ◽

1998 ◽

Vol 72 (7) ◽

pp. 5472-5480 ◽

Cited By ~ 50

Author(s):

Xu-Shan Wang ◽

Benjawan Khuntirat ◽

Keyun Qing ◽

Selvarangan Ponnazhagan ◽

Dagmar M. Kube ◽

...

Keyword(s):

Virus Type ◽

Viral Vector ◽

Nucleotide Sequencing ◽

Wild Type ◽

Helper Plasmid ◽

Adeno Associated Virus ◽

Recombinant Vector ◽

293 Cells ◽

Hairpin Structures

ABSTRACT The pSub201-pAAV/Ad plasmid cotransfection system was developed to eliminate homologous recombination which leads to generation of the wild-type (wt) adeno-associated virus type 2 (AAV) during recombinant vector production. The extent of contamination with wt AAV has been documented to range between 0.01 and 10%. However, the precise mechanism of generation of the contaminating wt AAV remains unclear. To characterize the wt AAV genomes, recombinant viral stocks were used to infect human 293 cells in the presence of adenovirus. Southern blot analyses of viral replicative DNA intermediates revealed that the contaminating AAV genomes were not authentic wt but rather wt AAV-like sequences derived from recombination between (i) AAV inverted terminal repeats (ITRs) in the recombinant plasmid and (ii) AAV sequences in the helper plasmid. Replicative AAV DNA fragments, isolated following amplification through four successive rounds of amplification in adenovirus-infected 293 cells, were molecularly cloned and subjected to nucleotide sequencing to identify the recombinant junctions. Following sequence analyses of 31 different ends of AAV-like genomes derived from two different recombinant vector stocks, we observed that all recombination events involved 10 nucleotides in the AAV D sequence distal to viral hairpin structures. We have recently documented that the first 10 nucleotides in the D sequence proximal to the AAV hairpin structures are essential for successful replication and encapsidation of the viral genome (X.-S. Wang et al., J. Virol. 71:3077–3082, 1997), and it was noteworthy that in each recombinant junction sequenced, the same 10 nucleotides were retained. We also observed that adenovirus ITRs in the helper plasmid were involved in illegitimate recombination with AAV ITRs, deletions of which significantly reduced the extent of wt AAV-like particles. Furthermore, the combined use of recombinant AAV plasmids lacking the distal 10 nucleotides in the D sequence and helper plasmids lacking the adenovirus ITRs led to complete elimination of replication-competent wt AAV-like particles in recombinant vector stocks. These strategies should be useful in producing clinical-grade AAV vectors suitable for human gene therapy.

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