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Under optimal conditions, the fidelity of DNA replication is extremely high. Indeed, it is estimated that, on average, only one error occurs for every 10 billion bases replicated. However, given that living organisms are continually subjected to a variety of endogenous and exogenous DNA-damaging agents, optimal conditions rarely prevail in vivo. While all organisms have evolved elaborate repair pathways to deal with such damage, the pathways rarely operate with 100% efficiency. Thus, the persisting DNA lesions are replicated, but with much lower fidelity than in undamaged DNA. Our aim is to understand the molecular mechanisms by which mutations are introduced into damaged DNA. The process, commonly referred to as translesion DNA synthesis (TLS), is facilitated by one or more members of the Y-family of DNA polymerases that are conserved from bacteria to humans. Based on phylogenetic relationships, Y-family polymerases may be broadly classified into five subfamilies; DinB-like (polIV/pol kappa-like) proteins are ubiquitous and found in all domains of life; in contrast, the Rev1-like, Rad30A (pol eta)-like, and Rad30B (pol iota)-like polymerases are found only in eukaryotes and the UmuC (polV)-like polymerases only in prokaryotes. We continue to investigate TLS in all three domains of life: bacteria, archaea, and eukaryotes.
Molecular breeding of DNA polymerases
The polymerase chain reaction (PCR) enables the detection, amplification, and interrogation of DNA sequences from minute starting quantities down to single DNA molecules, enabling a wealth of applications in medicine and biology ranging from clinical diagnostics, prognostics, forensics, to molecular genetics and 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. Indeed, a plethora of substances strongly inhibit polymerase activity and limit the use of the PCR in samples that contain such substances. Attempts have been made to mitigate the inhibitory effect on polymerase activity by increasing the concentration of the polymerase or by inclusing various additives. However, any anti-inhibitory effect is often sample-specific and is not always sufficient to ensure optimal PCR efficiency. In a collaborative study with Philipp Holliger, 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 the DNA polymerases of 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 the 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.
Identification of uracil residues in human immunoglobulin genes
After encountering foreign antigen, human B cells diversify their immunoglobulin genes by somatic hypermutation (SHM), gene conversion (GC) and class-switch recombination (CSR). SHM introduces mutations into variable region genes, and the mutant proteins are then selected by antigen to cause affinity maturation. Genomic mutagenesis is initiated by the activation-induced deaminase (AID) enzyme. AID is a member of the APOBEC family of polynucleotide deaminases that catalyze the conversion of cytosine to uracil in RNA and DNA. On the basis of its sequence similarity to the RNA–editing enzyme APOBEC1, it was initially thought that AID functions as a RNA deaminase. However, examination of AID activity in Escherichia coli and biochemical characterization of the AID protein has supported the idea that it deaminates DNA rather than RNA substrates. While most data support DNA deamination, there been is no physical evidence of uracil residues in immunoglobulin genes. In collaboration with Patricia Gearhart, we demonstrated the presence of uracils in DNA by determining the sensitivity of DNA to digestion with uracil DNA glycosylase (UNG) and abasic endonuclease (APE). Indeed, we identified uracil residues in the variable and switch regions of human immunoglobulin genes, using several different methods of detection. 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 provided the first direct evidence supporting the model that AID functions by deaminating cytosine residues in DNA, rather than in RNA.