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ABSTRACT: MRE11-RAD50 is a highly conserved multifunctional DNA repair factor. Here, we show that MRE11-RAD50 cleaves the covalent 3'-phosphotyrosyl-DNA bonds that join topoisomerase 1 (Top1) to the DNA backbone and that are the hallmark of damage caused by Top1 poisons such as camptothecin. Cleavage generates a 3'-phosphate DNA end that MRE11-RAD50 can resect in an ATP-regulated reaction, to produce a 3'-hydroxyl that can prime repair synthesis. The 3'-phosphotyrosyl cleavage activity maps to the MRE11 active site. These results define a new activity of MRE11 and distinguish MRE11-RAD50 functions in repair of Top1-DNA complexes and double-strand breaks.
Journal of Biological Chemistry 12/2011; 286(52):44945-51. · 4.77 Impact Factor
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ABSTRACT: APOBEC3G (A3G) restricts HIV-1 infection by catalyzing processive C --> U deaminations on single-stranded DNA (ssDNA) with marked 3' --> 5' deamination polarity. Here we show that A3G exists in oligomeric states whose composition is dictated primarily by interactions with DNA, with salt playing an important, yet secondary, role. Directional deaminations correlate with the presence of dimers, tetramers, and larger oligomers observed by atomic force microscopy, and random deaminations appear to correlate mainly with monomers. The presence of a 30-nt weakly deaminated "dead" zone located at the 3'-ssDNA end implies the presence of a preferred asymmetric direction for A3G catalysis. Single turnover reaction rates reveal a salt-dependent inhibition of C deamination toward the 3'-ssDNA region, offering a molecular basis underlying A3G deamination polarity. Presteady state analysis demonstrates rapid diffusion-limited A3G-ssDNA binding, a slower salt-dependent conformational change, possibly indicative of DNA wrapping, and long (5-15 min) protein-DNA complex lifetimes. We suggest that diverse A3G oligomerization modes contribute to the human immunodeficiency virus, type 1, proviral DNA mutational bias.
Journal of Biological Chemistry 06/2008; 283(20):13780-91. · 4.77 Impact Factor
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ABSTRACT: Hypermutation and class switch recombination of immunoglobulin genes are antigen-activated mechanisms triggered by AID, a cytidine deaminase. AID deaminates cytidine residues in the DNA of the variable and the switch regions of the immunoglobulin locus. The resulting uracil induces error-prone DNA synthesis in the case of hypermutation or DNA breaks that activate non-homologous recombination in the case of class switch recombination. In vitro studies have demonstrated that AID deaminates single-stranded but not double-stranded substrates unless AID is in a complex with RPA and the substrate is actively undergoing transcription. However, it is not clear whether AID deaminates its substrates primarily as a monomer or as a higher order oligomer. To examine the oligomerization state of AID alone and in the presence of single-stranded DNA substrates of various structures, including loops embedded in double-stranded DNA, we used atomic force microscopy (AFM) to visualize AID protein alone or in complex with DNA. Surprisingly, AFM results indicate that most AID molecules exist as a monomer and that it binds single-stranded DNA substrates as a monomer at concentrations where efficient deamination of single-stranded DNA substrates occur. The rate of deamination, under conditions of excess and limiting protein, also imply that AID can deaminate single-stranded substrates as a monomer. These results imply that non-phosphorylated AID is catalytically active as a monomer on single-stranded DNA in vitro, including single-stranded DNA found in loops similar to those transiently formed in the immunoglobulin switch regions during transcription.
DNA Repair 02/2008; 7(1):77-87. · 4.14 Impact Factor
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ABSTRACT: MutL alpha, the heterodimeric eukaryotic MutL homolog, is required for DNA mismatch repair (MMR) in vivo. It has been suggested that conformational changes, modulated by adenine nucleotides, mediate the interactions of MutL alpha with other proteins in the MMR pathway, coordinating the recognition of DNA mismatches by MutS alpha and the activation of MutL alpha with the downstream events that lead to repair. Thus far, the only evidence for these conformational changes has come from X-ray crystallography of isolated domains, indirect biochemical analyses, and comparison to other members of the GHL ATPase family to which MutL alpha belongs. Using atomic force microscopy (AFM), coupled with biochemical techniques, we demonstrate that adenine nucleotides induce large asymmetric conformational changes in full-length yeast and human MutL alpha and that these changes are associated with significant increases in secondary structure. These data reveal an ATPase cycle in which sequential nucleotide binding, hydrolysis, and release modulate the conformational states of MutL alpha.
Molecular Cell 02/2008; 29(1):112-21. · 14.18 Impact Factor
