THE HUMAN DNA REPAIR PROTEOME
In the last 15 years, we have accumulated over 50 genes that have demonstrated or consensus functions associated with human DNA repair. These genes/constructs have been cloned, completely sequenced to verify their integrity, and include the human mitotic MMR genes hMSH2, hMSH3, hMSH6, hMLH1, hMLH3, hPMS1 and hPMS2; the meiotic MutS homologs hMSH4 and hMSH5; the RecA homologs, hRAD51, hRAD51B, hRAD51C, hRAD51D, XRCC2, XRCC3, and hDMC1; the nucleotide excision repair and Xeroderma Pigmentosum genes XPA (hRAD14), XPB, XPD (hRAD3), XPF/ERCC4 (hRAD1), XPG (hRAD2), and ERCC1 (hRAD10); the glycosylases TDG, MBD4 and OGG1; the breast cancer susceptibility genes BRCA1 (its retinue BARD1 and BAP1) and BRCA2; the proteosomal components S3, S6, and DSS1; the helicases responsible for Bloom's Syndrome (BLM1) and Werner's Syndrome (WRN1); the mutagenic translesion and homologous recombination DNA synthesis polymerase complex consisting of hREV1, hREV3 and hREV7; the recombination/repair associated proteins ataxia telangiectasia mutated (ATM); the flap endonuclease (FEN1) and the hExoI exonuclease; the RAD54 helicase; the tripeptide single-stranded binding complex RPA (RPA1, RPA2 and RPA3); one of the human RAD52 homologs; and the seven known human Sir2 proteins (SIRT 1-7). We have developed a four-vector system with identical multiple cloning sites within which DNA repair genes may be inserted simultaneously: A GST-fusion vector (pGEX-SG1), an inducible bacterial vector (pET24d/29a), and the baculovirus vectors (pFastBacDual or pAcSG2). This multivector system provides a maximum of biochemical flexibility. Antibodies may be generated from GST-fusion peptide(s). Protein-protein interaction may be examined by immunoprecipitation or precipitation of with GST-fusion peptides followed by MassSpec analysis. Finally, the baculovirus vectors allow insect cell overexpression either as single proteins, co-infected proteins, or dual-expressed proteins. In addition, we have accumulated an enormous variety of useful reagents to study these DNA repair components (specific primers for PCR, cDNA sequencing, genomic sequencing; specific antibodies; gene fragments; peptide fragments). Most of our human antibody reagents cross-react with the corresponding mouse proteins.
The power of our collection of human DNA repair genes is illustrated by our as yet unpublished studies of simple protein interactions (Figure 7). A reciprocal interaction (reversing the protein bait) is required for us to declare a specific association. We have also begun to examine the dependence of adenosine nucleotide binding on protein-protein interactions (note green in Figure 7). The system may also be used to interrogate the specific peptide interactions regions of the DNA repair proteins as illustrated for the mitotic MSH and MLH proteins for ExoI. Our protein interaction technology has been greatly aided by the development of a high-throughput MALDI-ToF and LC/MS/MS(LTQ) MassSpec core facility.
PROJECTS (in collaboration with Dr. Christoph Schmutte)
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