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#double-strand breaks

7 public questions tagged with this topic.

Transgenic Plant -2

ZFNs induce genome modification by creating:

Zinc finger nucleases induce targeted genome modification by introducing double-strand breaks which are highly recombinogenic lesions activating cellular DNA damage response. After FokI dimer cleavage within spacer producing 5' overhangs, MRN complex Mre11-Rad50-Nbs1 senses ends, recruits ATM kinase phosphorylating H2AX, mediators 53BP1, initiating cell-cycle checkpoint. Breaks must be repaired to avoid apoptosis. In absence of donor, classical NHEJ mediated by Ku70/Ku80 heterodimer binding ends, DNA-PKcs recruitment, Artemis processing and Lig4-XRCC4 ligation rejoins ends frequently introduci

Ref: Jasin Rothstein CSH Perspect 2013 DSB repair; Carroll Genetics 2011 ZFN breaks DSB.

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Cell Cycle Regulation

Which DNA repair pathway is activated by ATM after detecting double-strand breaks?

Double-strand breaks present challenging repair choice between rapid but error-prone non-homologous end joining and accurate homologous recombination available only when sister chromatid present. ATM kinase activated by MRN complex phosphorylates H2AX, creating gamma-H2AX domains that recruit mediator 53BP1, which in turn favors end joining by blocking resection. In G1 phase when homologous template absent, ATM-53BP1 axis directs breaks toward classical non-homologous end joining pathway that directly ligates ends after minimal processing by Ku70/Ku80 heterodimer, DNA-PKcs, and XRCC4-DNA ligas

Ref: Marechal & Zou, Genes Dev 2013, DNA Damage Response; Scully et al., Cell 2019, DSB Repair Choice.

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Cell Cycle Regulation

Which kinase is activated in response to double-strand DNA breaks?

DNA double-strand breaks are among most deleterious lesions, potentially generating chromosomal translocations if unrepaired. Detection relies on sensor complex MRN composed of Mre11, Rad50, and Nbs1 that binds broken ends and recruits serine/threonine kinase ATM, ataxia telangiectasia mutated, member of PI3K-like kinase family. ATM exists as inactive dimer; upon recruitment and interaction with Nbs1 C-terminus, it autophosphorylates at serine 1981, dissociates into active monomers, and phosphorylates hundreds of targets including histone variant H2AX at serine 139 forming gamma-H2AX foci that

Ref: Abraham, Genes Dev 2001, ATM and ATR Kinases; Shiloh & Ziv, Nature Rev Mol Cell Biol 2013, ATM Signaling.

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Repair Pathway

Double-strand breaks are repaired error-free by

Double-strand break is most cytotoxic lesion resolved by two major pathways. Non-homologous end joining rapidly ligates ends but can delete nucleotides. Homology-directed repair, also termed homologous recombination repair or HDR, uses sister chromatid present in late S and G2 phases as faithful template. After resection by MRN complex CtIP, Rad51 catalyzes strand invasion forming D-loop, polymerase delta extends using homologous sequence, Holliday junctions resolved. Because homologous donor retains exact original sequence, repair restores information without errors, essential for maintaining

Ref: Lodish Molecular Cell Biology 9th ed. Chapter 12: HDR error-free DSB repair; Alberts Ch.5 Homologous recombination precise repair

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Recombination

Non-homologous end joining (NHEJ) is

Non-homologous end joining ligates broken chromosome ends without requiring homologous donor template, acting throughout cell cycle especially G1. Core factors Ku70/Ku80 anchor DNA ends, DNA-PKcs protects, Artemis trims, polymerases mu/lambda add nucleotides, and Lig4-XRCC4 seals ligation. Since processing removes or adds bases before joining, small insertions or deletions often arise at junction, causing frameshifts and mutations. This error-prone nature distinguishes it from homologous recombination which copies intact template accurately, but affords rapid survival when sister chromatid una

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 5: NHEJ as Error-Prone End Joining Mechanism

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Gene Tar getting

Cas9 endonuclease cuts the DNA and creates:

Cas9 is dual nuclease utilizing HNH domain to cut target strand complementary to guide RNA and RuvC domain to cut non-target strand simultaneously. Both nicks occur three base pairs upstream of PAM on opposite strands, generating predominantly blunt ended double strand breaks without single stranded overhangs. Such blunt breaks are predominantly repaired by non-homologous end joining causing small indels, or by homology directed repair if donor template provided. Blunt nature facilitates efficient ligation mediated insertion and predictable editing outcomes in genome engineering laboratories w

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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