Concatenation of Transgenic DNA: Random or Orchestrated?

Generation of transgenic organisms by pronuclear microinjection has become a routine procedure. However, while the process of DNA integration in the genome is well understood, we still do not know much about the recombination between transgene molecules that happens in the first moments after DNA injection. Most of the time, injected molecules are joined together in head-to-tail tandem repeats—the so-called concatemers. In this review, we focused on the possible concatenation mechanisms and how they could be studied with genetic reporters tracking individual copies in concatemers. We also discuss various features of concatemers, including palindromic junctions and repeat-induced gene silencing (RIGS). Finally, we speculate how cooperation of DNA repair pathways creates a multicopy concatenated insert.

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Disintegration promotes proto-spacer integration by the Cas1-Cas2 complex

10.1101/2020.12.21.423798 ◽

2020 ◽

Author(s):

Chien-Hui Ma ◽

Kamyab Javanmardi ◽

Ilya J. Finkelstein ◽

Makkuni Jayaram

Keyword(s):

Dna Repair ◽

Streptococcus Pyogenes ◽

Target Site ◽

Dna Transposition ◽

Insertion Event ◽

Dna Integration ◽

Biochemical Investigation ◽

Target Sites ◽

Repair Pathways ◽

Integration Event

Abstract‘Disintegration’—the reversal of transposon DNA integration at a target site—is regarded as an abortive off-pathway reaction. Here we challenge this view with a biochemical investigation of the mechanism of protospacer insertion by the Streptococcus pyogenes Cas1-Cas2 complex, which is mechanistically analogous to DNA transposition. In supercoiled target sites, the predominant outcome is the disintegration of one-ended insertions that fail to complete the second integration event. In linear target sites, one-ended insertions far outnumber complete proto-spacer insertions. The second insertion event is most often accompanied by disintegration of the first, mediated either by the 3’-hydroxyl exposed during integration or by water. One-ended integration intermediates may mature into complete spacer insertions via DNA repair pathways that are also involved in transposon mobility. We propose that disintegration-promoted integration is functionally important in the adaptive phase of CRISPR-mediated bacterial immunity, and perhaps in other analogous transposition reactions.

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Disintegration promotes protospacer integration by the Cas1-Cas2 complex

eLife ◽

10.7554/elife.65763 ◽

2021 ◽

Vol 10 ◽

Author(s):

Chien-Hui Ma ◽

Kamyab Javanmardi ◽

Ilya J Finkelstein ◽

Makkuni Jayaram

Keyword(s):

Dna Repair ◽

Streptococcus Pyogenes ◽

Target Site ◽

Dna Transposition ◽

Insertion Event ◽

Dna Integration ◽

Biochemical Investigation ◽

Target Sites ◽

Repair Pathways ◽

Integration Event

‘Disintegration’—the reversal of transposon DNA integration at a target site—is regarded as an abortive off-pathway reaction. Here, we challenge this view with a biochemical investigation of the mechanism of protospacer insertion, which is mechanistically analogous to DNA transposition, by the Streptococcus pyogenes Cas1-Cas2 complex. In supercoiled target sites, the predominant outcome is the disintegration of one-ended insertions that fail to complete the second integration event. In linear target sites, one-ended insertions far outnumber complete protospacer insertions. The second insertion event is most often accompanied by the disintegration of the first, mediated either by the 3′-hydroxyl exposed during integration or by water. One-ended integration intermediates may mature into complete spacer insertions via DNA repair pathways that are also involved in transposon mobility. We propose that disintegration-promoted integration is functionally important in the adaptive phase of CRISPR-mediated bacterial immunity, and perhaps in other analogous transposition reactions.

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Faculty Opinions recommendation of T-DNA integration in plants results from polymerase-θ-mediated DNA repair.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726907398.793525698 ◽

2016 ◽

Author(s):

Gene Nester

Keyword(s):

Dna Repair ◽

Dna Integration

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Histone methylation can either promote or reduce cellular radiosensitivity by regulating DNA repair pathways

Mutation Research/Reviews in Mutation Research ◽

10.1016/j.mrrev.2020.108362 ◽

2021 ◽

Vol 787 ◽

pp. 108362

Author(s):

Yuchuan Zhou ◽

Chunlin Shao

Keyword(s):

Dna Repair ◽

Histone Methylation ◽

Cellular Radiosensitivity ◽

Repair Pathways

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Targeting DNA Repair and Chromatin Crosstalk in Cancer Therapy

Cancers ◽

10.3390/cancers13030381 ◽

2021 ◽

Vol 13 (3) ◽

pp. 381

Author(s):

Danielle P. Johnson ◽

Mahesh B. Chandrasekharan ◽

Marie Dutreix ◽

Srividya Bhaskara

Keyword(s):

Dna Repair ◽

Cancer Therapy ◽

Therapeutic Intervention ◽

Synthetic Lethality ◽

Checkpoint Proteins ◽

Cellular Processes ◽

Repair Pathways ◽

Made In

Aberrant DNA repair pathways that underlie developmental diseases and cancers are potential targets for therapeutic intervention. Targeting DNA repair signal effectors, modulators and checkpoint proteins, and utilizing the synthetic lethality phenomena has led to seminal discoveries. Efforts to efficiently translate the basic findings to the clinic are currently underway. Chromatin modulation is an integral part of DNA repair cascades and an emerging field of investigation. Here, we discuss some of the key advancements made in DNA repair-based therapeutics and what is known regarding crosstalk between chromatin and repair pathways during various cellular processes, with an emphasis on cancer.

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