Repair Pathway

Repair Pathway

Stalled RNA polymerase during TCR recruits repair via

In bacterial transcription-coupled repair, stalled RNA polymerase at bulky lesion creates roadblock preventing repair access. Transcription-repair coupling factor Mfd, also known as TRCF, ATPase of superfamily 2, binds upstream DNA and uses ATP-driven translocation to displace stalled polymerase. Mfd contains UvrB homology module that directly recruits nucleotide excision repair machinery UvrA2B complex to damage site. By dissociating polymerase and loading Uvr proteins, Mfd couples transcription state to excision repair. MutS acts in mismatch repair, UvrD in unwinding, RecA in recombination, not transcription repair coupling, highlighting Mfd specificity.

Ref: NCBI Bookshelf TRCF Mfd function in TCR; Alberts et al. Chapter 6: Bacterial TCR mechanism; ScienceDirect Mfd UvrA recruitment

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Repair Pathway

Transcription-coupled NER repairs damage on

Transcription-coupled nucleotide excision repair is subpathway accelerating removal of bulky helix-distorting lesions that block elongating RNA polymerase. When RNA polymerase II stalls at cyclobutane pyrimidine dimer or 6-4 photoproduct located on transcribed template strand, stalled complex is recognized by CSB and CSA ubiquitin ligase recruiting TFIIH and core excision factors. Repair focused exclusively on template strand because non-transcribed strand lesions do not impede polymerase translocation. This strand-specific prioritization ensures rapidly transcribed essential genes restored preferentially, explaining clinical features of Cockayne syndrome defective in this pathway versus global genome repair.

Ref: Watson Molecular Biology of Gene 7th ed. Chapter 10: TCR repairs template strand lesions; Alberts Ch.5 Transcription-coupled NER

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Repair Pathway

Final ligation in NHEJ is done by

Final sealing of processed DNA ends in classical non-homologous end joining performed by DNA ligase IV, dedicated ligase distinct from replication ligase I and base excision ligase III. Ligase IV forms stable obligate complex with XRCC4 scaffold and accessory factor XLF/Cernunnos plus PAXX, enhancing adenylation and end alignment. After Ku and DNA-PKcs orchestrate end processing, XRCC4-Ligase IV complex catalyzes ATP-dependent phosphodiester bond formation joining broken termini. Patient mutations in LIG4 cause immunodeficiency and radiosensitivity, highlighting essential role in lymphocyte V(D)J recombination and general double-strand break repair pathway completion.

Ref: Lodish Chapter 11: NHEJ ligation by Ligase IV XRCC4 complex; J Biol Chem 2018 Mechanism of Ligase IV end joining

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Repair Pathway

Broken DNA ends in NHEJ are recognized by

Ku heterodimer composed of 70 kDa and 80 kDa subunits forms preformed ring with central aperture approximately 30 angstroms accommodating double-stranded DNA ends without sequence specificity. Abundant nuclear protein diffuses, threading onto broken terminus within seconds, protecting end from nucleolytic degradation and acting as scaffold for non-homologous end joining machinery. Ku recruits DNA-dependent protein kinase catalytic subunit, Artemis nuclease, polymerases mu and lambda, and XRCC4-Ligase IV complex. By synapsing two Ku-bound ends, it holds broken chromosome together. Rapid binding also antagonizes resection, steering repair away from homologous recombination.

Ref: Alberts Molecular Biology of Cell Chapter 5: Ku70/Ku80 end recognition in NHEJ; Nature Struct Mol Biol 2017 Ku-DNA complex

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Repair Pathway

NHEJ is predominant in which cell cycle phase?

Pathway choice for double-strand break repair depends strongly on cell cycle stage and resection control. In G0 quiescence and G1 gap where sister chromatids absent, extensive 5' resection blocked by 53BP1-RIF1-Shieldin complex and Ku end-binding factor prevents homology search. Therefore non-homologous end joining dominates, initiated by Ku70/Ku80 rapidly capping ends and recruiting DNA-PKcs and ligase IV. In S/G2, CDK phosphorylation activates CtIP enabling resection favoring homologous recombination. Thus NHEJ predominance in G0/G1 reflects availability of template and protective mechanisms limiting homologous recombination when homolog unavailable.

Ref: Berg Biochemistry Chapter 28: DSB repair cell cycle choice; NCBI Bookshelf NHEJ in G1 predominance regulation

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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 genome stability and enabling precise genome editing applications using donor templates.

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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Repair Pathway

RecA activates SOS response by inducing cleavage of

RecA forms helical nucleoprotein filament on single-stranded DNA generated at lesion-stalled forks, with ATP stabilizing active extended conformation termed RecA*. Activated filament functions as coprotease enhancing intrinsic autoproteolytic activity of LexA, UmuD, and phage lambda repressor. LexA self-cleavage occurs between alanine 84 and glycine 85 within flexible linker connecting N-terminal DNA-binding domain and C-terminal dimerization domain. Cleavage splits repressor, rendering fragments unable to dimerize and bind SOS box. Consequently operons derepress without RecA directly cutting LexA. Signal transduction links recombinational sensor to transcriptional response essential for stress adaptation.

Ref: PNAS 2021 Cryo-EM RecA-LexA filament structure autocleavage; Alberts Ch.5 RecA coprotease mechanism SOS activation

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Repair Pathway

SOS response is regulated by

SOS response encompasses global induction of more than forty DNA repair and tolerance genes triggered by extensive single-stranded DNA at stalled replication forks. Central negative regulator is LexA repressor, homodimer binding SOS boxes with consensus CTGTN8ACAG in promoter regions, repressing transcription including own lexA gene and recA, uvr, umuDC operons. Under steady growth, LexA maintains low expression. After DNA damage, activated RecA nucleofilament stimulates LexA autocleavage at Ala84-Gly85, causing dissociation from operators and derepression. Temporal induction occurs as LexA affinity varies across promoters, coordinating error-free and error-prone repair sequentially.

Ref: Alberts Molecular Biology of Cell, 7th ed., Chapter 5: SOS regulon LexA repressor control; Scielo 2020 SOS regulation E coli

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Repair Pathway

DNA Pol IV and V belong to which polymerase family?

Escherichia coli DNA polymerases IV encoded by dinB and V encoded by umuD'2C belong to Y-family of translesion polymerases, distinct from canonical A-family, B-family replicases, and X-family repair enzymes. Y-family members share unique structural features including little finger domain, unusually open solvent-accessible active site that accommodates bulky lesions such as benzo[a]pyrene adducts and thymine dimers, and absence of proofreading domain. They display low processivity, synthesizing only few nucleotides per binding event. Evolutionary conservation includes human Pol eta, iota, kappa. This classification explains low fidelity and lesion bypass specialization.

Ref: Lehninger Principles of Biochemistry, 8th ed., Chapter 27: Y-family polymerase structural features; EMBO J 1999 Pol IV V identification

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Repair Pathway

Translesion DNA synthesis is

Translesion DNA synthesis represents damage tolerance mechanism allowing replication fork to bypass bulky adducts, pyrimidine dimers, cisplatin crosslinks, or apurinic sites that block high-fidelity replicative polymerases. Specialized Y-family polymerases Pol IV and Pol V possess capacious active sites accommodating distorted base pairs but lack 3' to 5' proofreading exonuclease and exhibit relaxed Watson-Crick selection. As result, they insert nucleotides opposite lesions with high misincorporation rates, generating targeted and untargeted mutations in SOS response. Though mutagenic, TLS prevents double-strand breaks caused by fork collapse and enhances survival under genotoxic stress.

Ref: Friedberg et al., DNA Repair and Mutagenesis, 2nd ed., Chapter 15: TLS error-prone bypass mechanism; Alberts Ch.5 SOS translesion polymerases

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Repair Pathway

Mismatch repair in eukaryotes lacks

Eukaryotic mismatch repair retains conserved MutS homologs MSH2/MSH6 for mismatch recognition and MutL homologs MLH1/PMS2 for coordination, but lacks MutH ortholog entirely. Strand discrimination does not depend on Dam methylation, absent in eukaryotes. Instead, naturally occurring nicks associated with lagging strand Okazaki fragment termini, leading strand replication discontinuities, or PCNA-bound MutL endonuclease activity create entry points for EXO1 exonuclease. MutLα itself displays latent endonuclease regulated by PCNA and ATP. Therefore, eukaryotic system achieves directionality without methylation-directed MutH cleavage, representing mechanistic divergence.

Ref: NCBI Bookshelf DNA Mismatch Repair in Eukaryotes No MutH; Lodish Molecular Cell Biology 9th ed. Chapter 11 Eukaryotic MMR differences

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Repair Pathway

Mismatch excision requires which helicase?

Following MutH nicking of daughter strand at hemimethylated GATC, intervening DNA between nick and mismatch must be unwound and removed. Helicase II, named UvrD, is recruited through direct protein interaction with MutL. Using ATP hydrolysis, UvrD translocates 3' to 5' direction, unwinding duplex flanking mismatch region. Single-stranded DNA binding protein stabilizes exposed strands preventing reannealing. Subsequently, appropriate exonucleases degrade unwound strand: 3' to 5' exonucleases if nick is upstream, 5' to 3' RecJ if nick downstream. DNA polymerase III then fills gap accurately.

Ref: Watson et al., Molecular Biology of the Gene, 7th ed., Chapter 10: MMR excision helicase UvrD role; Alberts Ch.5 Exonucleolytic removal

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