Cell Biology & Molecular Genetics

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Author: search.filters.author.Li, Zhuo

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    A nuclear magnetic resonance based approach to accurate functional annotation of putative enzymes in the methanogen Methanosarcina acetivorans
    (Springer Nature, 2011-06-15) Chen, Yihong; Apolinario, Ethel; Brachova, Libuse; Kelman, Zvi; Li, Zhuo; Nikolau, Basil J; Showman, Lucas; Sowers, Kevin; Orban, John
    Correct annotation of function is essential if one is to take full advantage of the vast amounts of genomic sequence data. The accuracy of sequence-based functional annotations is often variable, particularly if the sequence homology to a known function is low. Indeed recent work has shown that even proteins with very high sequence identity can have different folds and functions, and therefore caution is needed in assigning functions by sequence homology in the absence of experimental validation. Experimental methods are therefore needed to efficiently evaluate annotations in a way that complements current high throughput technologies. Here, we describe the use of nuclear magnetic resonance (NMR)-based ligand screening as a tool for testing functional assignments of putative enzymes that may be of variable reliability. The target genes for this study are putative enzymes from the methanogenic archaeon Methanosarcina acetivorans (MA) that have been selected after manual genome re-annotation and demonstrate detectable in vivo expression at the level of the transcriptome. The experimental approach begins with heterologous E. coli expression and purification of individual MA gene products. An NMR-based ligand screen of the purified protein then identifies possible substrates or products from a library of candidate compounds chosen from the putative pathway and other related pathways. These data are used to determine if the current sequence-based annotation is likely to be correct. For a number of case studies, additional experiments (such as in vivo genetic complementation) were performed to determine function so that the reliability of the NMR screen could be independently assessed. In all examples studied, the NMR screen was indicative of whether the functional annotation was correct. Thus, the case studies described demonstrate that NMR-based ligand screening is an effective and rapid tool for confirming or negating the annotated gene function of putative enzymes. In particular, no protein-specific assay needs to be developed, which makes the approach broadly applicable for validating putative functions using an automated pipeline strategy.
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    ARCHAEAL DNA REPLICATION PROTEINS: MEMBERS AND FUNCTIONS
    (2013) Li, Zhuo; Kelman, Zvi; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The mechanism of DNA replication in archaea, the third domain of life, has been studied for more than two decades using biochemical, structural and bioinformatic approaches. Historically, many of the proteins that participate in archaeal replication were identified via similarity to enzymes needed for DNA replication in bacteria and eukarya. This study uses a different approach to identify new factors that may be involved in replication. Genetic tools developed for the thermophilic archaeon Thermococcus kodakarensis were used to identify new replication factors that could not be recognized through in silico methods. First, a network of proteins that may participate in replication was identified using in vivo tagging of known replication enzymes. Following affinity purification the proteins that co-purified with the tagged enzymes were identified using mass spectrometry. This study describes the identification of a number of new putative replication factors. Next, the biochemical properties of two proteins identified in the screen were characterized. One, the product of gene TK1525, was identified via its interaction with the GINS complex. This protein was predicted to be an archaeal homologue of the bacterial RecJ nuclease. It was found that the protein is a processive, manganese-dependent, single strand DNA-specific exonuclease. The protein was designated GAN for GINS-associated nuclease. GAN forms a complex with GINS and also interacts with the archaeal-specific DNA polymerase D in vivo. Subsequent bioinformatic analysis suggested that GAN may be the archaeal homologue of the eukaryotic Cdc45 protein. The second protein characterized is the product of TK0808. This protein was identified via its interactions with proliferating cell nuclear antigen (PCNA). The protein, upon binding to PCNA, inhibits PCNA-dependent activities. The protein was therefore designated TIP for Thermococcales inhibitor of PCNA. While most proteins that interact with PCNA do so via a PCNA-interacting peptide (PIP) motif that interacts with the inter domain connecting loop (IDCL) on PCNA, TIP neither contains the canonical PIP motif nor interacts with PCNA via the IDCL. These findings suggest a new mechanism for PCNA binding and suggest a new mechanism to regulate PCNA-dependent activities.