Uracil DNA N-Glycosylase Promotes Assembly of Human Centromere Protein A

Uracil is removed from DNA by the conserved enzyme Uracil DNA N-glycosylase (UNG). Previously, we observed that inhibiting UNG in Xenopus egg extracts blocked assembly of CENP-A, a histone H3 variant. CENP-A is an essential protein in all species, since it is required for chromosome segregation during mitosis. Thus, the implication of UNG in CENP-A assembly implies that UNG would also be essential, but UNG mutants lacking catalytic activity are viable in all species. In this paper, we present evidence that UNG2 colocalizes with CENP-A and H2AX phosphorylation at centromeres in normally cycling cells. Reduction of UNG2 in human cells blocks CENP-A assembly, and results in reduced cell proliferation, associated with increased frequencies of mitotic abnormalities and rapid cell death. Overexpression of UNG2 induces high levels of CENP-A assembly in human cells. Using a multiphoton laser approach, we demonstrate that UNG2 is rapidly recruited to sites of DNA damage. Taken together, our data are consistent with a model in which the N-terminus of UNG2 interacts with the active site of the enzyme and with chromatin

Cdc9 DNA ligase

C-terminal domain of yeast PCNA is required for physical and functional interactions wit

Double-strand DNA breaks recruit the centromeric histone CENP-A

The histone H3 variant CENP-A is required for epigenetic specification of centromere identity through a loading mechanism independent of DNA sequence. Using multiphoton absorption and DNA cleavage at unique sites by I-SceI endonuclease, we demonstrate that CENP-A is rapidly recruited to double-strand breaks in DNA, along with three components (CENP-N, CENP-T, and CENP-U) associated with CENP-A at centromeres. The centromere-targeting domain of CENP-A is both necessary and sufficient for recruitment to double-strand breaks. CENP-A accumulation at DNA breaks is enhanced by active non-homologous end-joining but does not require DNA-PKcs or Ligase IV, and is independent of H2AX. Thus, induction of a double-strand break is sufficient to recruit CENP-A in human and mouse cells. Finally, since cell survival after radiation-induced DNA damage correlates with CENP-A expression level, we propose that CENP-A may have a function in DNA repair

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

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

Three Metal Ions Participate in the Reaction Catalyzed by T5 Flap Endonuclease*S⃞

Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a “two-metal-ion mechanism.” Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of >4 Å appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2+ or Mn2+). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal-dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions