Accumulation of XRCC1 and LIGIIIα at SSBs after local UV-irradiation in XPA-UVDE cells

<p><b>Copyright information:</b></p><p>Taken from "Translocation of XRCC1 and DNA ligase IIIα from centrosomes to chromosomes in response to DNA damage in mitotic human cells"</p><p>Nucleic Acids Research 2005;33(1):422-429.</p><p>Published online 14 Jan 2005</p><p>PMCID:PMC546168.</p><p>© 2005, the authors © </p> Co-localization of XRCC1 with LIGIIIα was identified by double immunolabeling. Two minutes after local UV-irradiation (20 J/m) cells were fixed with paraformaldehyde and co-stained with anti-XRCC1 antibody (red, upper row) and anti-LIGIIIα antibody (green, middle row); the column c is for XPA-UVDE cells treated with DIQ, an inhibitor of PARP, before UV-irradiation. Co-localization of XRCC1 with LIGIIIα appears yellow in overlay (bottom row)

Crystal structure of yPCNA complexed with a Cdc9 peptide encompassing the PIP box

<p><b>Copyright information:</b></p><p>Taken from "The C-terminal domain of yeast PCNA is required for physical and functional interactions with Cdc9 DNA ligase"</p><p></p><p>Nucleic Acids Research 2007;35(5):1624-1637.</p><p>Published online 18 Feb 2007</p><p>PMCID:PMC1865074.</p><p>© 2007 The Author(s).</p> () Cdc9 peptide sequence used for crystallization showing the secondary structure of residues observed in the co-crystal structure (bold black letters) in relation to disordered residues (gray letters) that had no observed electron density. The Cdc9 peptide is aligned with the PIP box and surrounding residues from human DNA ligase I (hDNA Lig I), yeast Rfc-1 (Rfc1), yeast FEN-1 (Rad27) and yeast Pol δ (Pol32). The starting and ending residue numbers, along with the total sequence length is shown for each protein. Key residues of the PIP box are denoted by asterisks (*) below the sequence. () Structure of the Cdc9 peptide:yPCNA complex shown as a trimer based on crystallographic symmetry. The yPCNA trimer is shown as a ribbon (subunits colored beige, blue and pink) with colored spheres marking the position of mutant residues in the pcna-79 (blue spheres) and pcna-90 (red spheres) mutants. The Cdc9 peptide (green) is bound to each of the subunits

Interaction of Cdc9 with wild-type and mutant versions of PCNA in the absence of DNA

<p><b>Copyright information:</b></p><p>Taken from "The C-terminal domain of yeast PCNA is required for physical and functional interactions with Cdc9 DNA ligase"</p><p></p><p>Nucleic Acids Research 2007;35(5):1624-1637.</p><p>Published online 18 Feb 2007</p><p>PMCID:PMC1865074.</p><p>© 2007 The Author(s).</p> (). Schematic of sensor chip used in surface plasmon resonance analysis of association of Cdc9 with PCNA. PCNA wild-type PCNA (P-W), pcna-79 (P-79) and pcna-90 (P-90) were immobilized to a CM5 chip and 100 nM of GST fused to either wild-type Cdc9 (Wt) or Cdc9 FFAA mutant (PIP box Mut) were passed over the chip. (). Sensorgram showing the association and disassociation curves of GST-Cdc9 wild-type (C-W) with PCNA wild-type (P-W/C-W), pcna79 (P-79/C-W) and pcna-90 (P-90/C-W) and of GST Cdc9 FFAA mutant (PIP box Mutant; C-M) with PCNA wild-type (P-W/C-M), pcna79 (P-79/C-M) and pcna-90 (P-90/C-M). () Pull-down assays. Lanes 1–3 represent 10% of input PCNA used in the pull-down assay. Purified PCNA (P-W), pcna-79 (P-79) and pcna-90 (P-90) (5 pmol of PCNA trimer) were incubated with glutathione sepharose beads liganded by 5 pmol of GST, GST–Cdc9 wild-type (GSTCdc9 Wt) or GST-Cdc9-PIP box mutant (GST Cdc9 PIP Mut). Proteins bound to GST beads (lanes 4–6), GST–Cdc9 Wt beads (lanes 7–9) and GST–Cdc9p PIP Mut beads (lanes 10–12) were separated by SDS-PAGE. PCNA (top panel) and GST-Cdc9p (bottom panel) were detected by immunoblotting with Cdc9 and PCNA antibodies, respectively

The Nucleotide Sequence, DNA Damage Location, and Protein Stoichiometry Influence the Base Excision Repair Outcome at CAG/CTG Repeats

Expansion of CAG/CTG repeats is the underlying cause of >14 genetic disorders, including Huntington’s disease (HD) and myotonic dystrophy. The mutational process is ongoing, with increases in repeat size enhancing the toxicity of the expansion in specific tissues. In many repeat diseases, the repeats exhibit high instability in the striatum, whereas instability is minimal in the cerebellum. We provide molecular insights into how base excision repair (BER) protein stoichiometry may contribute to the tissue-selective instability of CAG/CTG repeats by using specific repair assays. Oligonucleotide substrates with an abasic site were mixed with either reconstituted BER protein stoichiometries mimicking the levels present in HD mouse striatum or cerebellum, or with protein extracts prepared from HD mouse striatum or cerebellum. In both cases, the repair efficiency at CAG/CTG repeats and at control DNA sequences was markedly reduced under the striatal conditions, likely because of the lower level of APE1, FEN1, and LIG1. Damage located toward the 5′ end of the repeat tract was poorly repaired, with the accumulation of incompletely processed intermediates as compared to an AP lesion in the center or at the 3′ end of the repeats or within control sequences. Moreover, repair of lesions at the 5′ end of CAG or CTG repeats involved multinucleotide synthesis, particularly at the cerebellar stoichiometry, suggesting that long-patch BER processes lesions at sequences susceptible to hairpin formation. Our results show that the BER stoichiometry, nucleotide sequence, and DNA damage position modulate repair outcome and suggest that a suboptimal long-patch BER activity promotes CAG/CTG repeat instability

The Nucleotide Sequence, DNA Damage Location, and Protein Stoichiometry Influence the Base Excision Repair Outcome at CAG/CTG Repeats

Expansion of CAG/CTG repeats is the underlying cause of >14 genetic disorders, including Huntington’s disease (HD) and myotonic dystrophy. The mutational process is ongoing, with increases in repeat size enhancing the toxicity of the expansion in specific tissues. In many repeat diseases, the repeats exhibit high instability in the striatum, whereas instability is minimal in the cerebellum. We provide molecular insights into how base excision repair (BER) protein stoichiometry may contribute to the tissue-selective instability of CAG/CTG repeats by using specific repair assays. Oligonucleotide substrates with an abasic site were mixed with either reconstituted BER protein stoichiometries mimicking the levels present in HD mouse striatum or cerebellum, or with protein extracts prepared from HD mouse striatum or cerebellum. In both cases, the repair efficiency at CAG/CTG repeats and at control DNA sequences was markedly reduced under the striatal conditions, likely because of the lower level of APE1, FEN1, and LIG1. Damage located toward the 5′ end of the repeat tract was poorly repaired, with the accumulation of incompletely processed intermediates as compared to an AP lesion in the center or at the 3′ end of the repeats or within control sequences. Moreover, repair of lesions at the 5′ end of CAG or CTG repeats involved multinucleotide synthesis, particularly at the cerebellar stoichiometry, suggesting that long-patch BER processes lesions at sequences susceptible to hairpin formation. Our results show that the BER stoichiometry, nucleotide sequence, and DNA damage position modulate repair outcome and suggest that a suboptimal long-patch BER activity promotes CAG/CTG repeat instability

The Nucleotide Sequence, DNA Damage Location, and Protein Stoichiometry Influence the Base Excision Repair Outcome at CAG/CTG Repeats

Expansion of CAG/CTG repeats is the underlying cause of >14 genetic disorders, including Huntington’s disease (HD) and myotonic dystrophy. The mutational process is ongoing, with increases in repeat size enhancing the toxicity of the expansion in specific tissues. In many repeat diseases, the repeats exhibit high instability in the striatum, whereas instability is minimal in the cerebellum. We provide molecular insights into how base excision repair (BER) protein stoichiometry may contribute to the tissue-selective instability of CAG/CTG repeats by using specific repair assays. Oligonucleotide substrates with an abasic site were mixed with either reconstituted BER protein stoichiometries mimicking the levels present in HD mouse striatum or cerebellum, or with protein extracts prepared from HD mouse striatum or cerebellum. In both cases, the repair efficiency at CAG/CTG repeats and at control DNA sequences was markedly reduced under the striatal conditions, likely because of the lower level of APE1, FEN1, and LIG1. Damage located toward the 5′ end of the repeat tract was poorly repaired, with the accumulation of incompletely processed intermediates as compared to an AP lesion in the center or at the 3′ end of the repeats or within control sequences. Moreover, repair of lesions at the 5′ end of CAG or CTG repeats involved multinucleotide synthesis, particularly at the cerebellar stoichiometry, suggesting that long-patch BER processes lesions at sequences susceptible to hairpin formation. Our results show that the BER stoichiometry, nucleotide sequence, and DNA damage position modulate repair outcome and suggest that a suboptimal long-patch BER activity promotes CAG/CTG repeat instability