Project description:The mismatch repair (MMR) family is a highly conserved group of proteins that function in correcting base-base and insertion-deletion mismatches generated during DNA replication. To systematically investigate the mismatch repair pathway, we conducted a proteomic analysis and identified MMR-associated protein complexes using a tandem-affinity purification coupled with mass spectrometry (TAP-MS) method. In total, we identified 262 high-confidence candidate interaction proteins (HCIPs).
Project description:The mismatch repair (MMR) family is a highly conserved group of proteins that function in correcting base-base and insertion-deletion mismatches generated during DNA replication. To systematically investigate the mismatch repair pathway, we conducted a proteomic analysis and identified MMR-associated protein complexes using a tandem-affinity purification coupled with mass spectrometry (TAP-MS) method. In total, we identified 262 high-confidence candidate interaction proteins (HCIPs).
Project description:Eukaryotic mismatch-repair (MMR) proteins MutSalpha and MutLalpha couple recognition of base mismatches to strand-specific excision, initiated in vivo at growing 3' ends and 5' Okazaki-fragment ends or, in human nuclear extracts, at nicks in exogenous circular substrates. We addressed five biochemical questions relevant to coupling models. Excision remained fully efficient at DNA:MutSalpha ratios of nearly 1 to 1 at various mismatch-nick distances, suggesting a requirement for only one MutSalpha molecule per substrate. As the mismatch-nick DNA contour distance D in exogenous substrates increased from 0.26 to 0.98 kbp, initiation of excision in extracts decreased as D(-0.43) rather than the D(-1) to D(-2) predicted by some translocation or diffusion models. Virtually all excision was along the shorter (3'-5') nick-mismatch, even when the other (5'-3') path was less than twice as long. These observations argue against stochastically directed translocating/diffusing recognition complexes. The failure of mismatched DNA in trans to provoke excision of separate nicked homoduplexes argues against one-stage (concerted) triggering of excision initiation by recognition complexes acting through space. However, proteins associated with gapped DNA did appear to compete in trans with those in cis to mismatch-associated proteins. Thus, as in Escherichia coli, eukaryotic MMR may involve distinct initial-activation and excision-path-commitment stages.
Project description:Human monocyte-derived macrophages (MDM) serve as a model for resident alveolar macrophages (AM) in the human respiratory tract. mRNA-Seq analysis was used to profile the cellular transcriptome of MDM cells at multiple time points in response to infection with influenza A/California/04/09 (H1N1), A/Wyoming/03/03 (H3N2), and A/Vietnam/1203/04 (H5N1) HALo virus. The Influenza A/Vietnam/1203/04 (H5N1) HALo mutant virus is an attenuated H5N1 virus generated from wild-type Influenza A/Vietnam/1203/04 (H5N1) virus as described in Steel, J., et al. J Virol. 2009 Feb; 83(4):1742-53.
Project description:Human tracheobronchial epithelial (HTBE) cells are considered to serve as a good correlate of influenza virus infection in the human respiratory tract. mRNA-Seq analysis was used to profile the cellular transcriptome of HTBE cells at multiple time points in response to infection with influenza A/California/04/09 (H1N1), A/Wyoming/03/03 (H3N2), and A/Vietnam/1203/04 (H5N1) HALo virus. The Influenza A/Vietnam/1203/04 (H5N1) HALo mutant virus is an attenuated H5N1 virus generated from wild-type Influenza A/Vietnam/1203/04 (H5N1) virus as described in Steel, J., et al. J Virol. 2009 Feb; 83(4):1742-53.
Project description:Human tracheobronchial epithelial (HTBE) cells are considered to serve as a good correlate of influenza virus infection in the human respiratory tract. ChIP-Seq analysis was used to profile histone acetylation (H3K27ac) in HTBE cells at multiple time points in response to infection with influenza A/California/04/09 (H1N1), A/Wyoming/03/03 (H3N2), and A/Vietnam/1203/04 (H5N1) HALo virus. The Influenza A/Vietnam/1203/04 (H5N1) HALo mutant virus is an attenuated H5N1 virus generated from wild-type Influenza A/Vietnam/1203/04 (H5N1) virus as described in Steel, J., et al. J Virol. 2009 Feb; 83(4):1742-53.
Project description:Mismatched base pairs, produced by nucleotide misincorporation by DNA polymerase, are repaired by the mismatch repair (MMR) pathway to maintain genetic integrity. We have developed a method for the fluorescence detection of the cellular MMR ability. A mismatch, which would generate a stop codon in the mRNA transcript unless it was repaired, was introduced into the gene encoding the enhanced green fluorescent protein (EGFP) in an expression plasmid. When MMR-proficient HeLa cells were transformed with this plasmid, the production of active EGFP was observed by fluorescence microscopy. It was assumed that the nick required to initiate the MMR pathway was produced non-specifically in the cells. In contrast, fluorescence was not detected for three types of MMR-deficient cells, LoVo, HCT116, and DLD-1, transformed with the same plasmid. In addition, the expression of a red fluorescent protein gene was utilized to avoid false-negative results. This simple fluorescence method may improve the detection of repair defects, as a biomarker for cancer diagnosis and therapy.
Project description:Hi-C was used to profile changes in the genome structure of human primary cells at multiple time points in response to infection with active and UV-inactivated H5N1 influenza virus. Human tracheobronchial epithelial cells (HTBE) and monocyte-derived macrophages (MDM) were used. The Influenza A/Vietnam/1203/04 (H5N1) HALo mutant virus is an attenuated H5N1 virus generated from wild-type Influenza A/Vietnam/1203/04 (H5N1) virus as described in Steel, J., et al. J Virol. 2009 Feb; 83(4):1742-53.