DNA Replication, Repair, and Mutagenesis In Eukaryotic And Prokaryotic Cells

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Scientists within the Laboratory of Genomic Integrity (LGI) study the mechanisms by which mutations are introduced into DNA. These studies span the evolutionary spectrum and include studies in bacteria, archaea and eukaryotes In humans, Activation-Induced Deaminase (AID) initiates diversity of immunoglobulin genes through deamination of Cytosine to Uracil. However, it has yet to be conclusively determined if the deamination event occurs in DNA, or RNA. Although most data support DNA deamination, there been is no physical evidence of Uracil residues in immunoglobulin genes. In a collaborative study with Patricia Gearhart (NIA/NIH), we demonstrated their presence by determining the sensitivity of DNA to digestion with Uracil DNA glycosylase (UNG) and abasic endonuclease. Using several different methods of detection, we identified Uracil residues in the variable and switch regions of human immunoglobulin genes. Uracil residues were generated within 24 h of B cell stimulation, were present on both DNA strands and were found to replace mainly Cytosine bases. Our data therefore provides the first direct evidence supporting the model that AID functions by deaminating Cytosine residues in DNA, rather than in RNA. DNA polymerases from Archaea are often thermostable and have been used for several decades in the polymerase chain reaction (PCR). PCR enables the detection, amplification and interrogation of DNA sequences from minute starting quantities, down to single DNA molecules. This has enabled a wealth of applications in medicine and biology ranging from clinical diagnostics, prognostics, and forensics, to molecular genetics including molecular archaeology and palaeobiology. However, the utility of PCR assays and the recovery of amplicons from such specimens can be greatly hindered, or even abrogated, by the presence of potent inhibitors. In a collaborative study with Phillip Holliger (MRC, Cambridge UK), we used molecular breeding and compartmentalized self-replication (CSR) of eight different Thermus DNA polymerase orthologs to engineer novel DNA polymerases with a broad resistance to complex environmental inhibitors. One such enzyme, called 2D9, was a chimeric polymerase comprising sequence elements derived from DNA polymerases from Thermus aquaticus, Thermus oshimai, Thermus thermophilus and Thermus brockianus. Remarkably, the 2D9 polymerase displayed a striking resistance to a broad spectrum of complex inhibitors of highly divergent composition including humic acid, bone dust, coprolite, peat extract, clay-rich soil, cave sediment and tar. We believe that 2D9 chimeric polymerase promises to have utility in PCR-based applications in a wide range of scientific fields including palaeobiology, archaeology, conservation biology, forensic and historic medicine.