DNA base excision repair at high temperatures

In all organisms, DNA bases are subject to chemical modifications causing cell death and/or mutagenesis if left unrepaired. Such damage can be enzymatically repaired by the base excision repair pathway (BER), which is initiated by removal of the modified base by a DNA glycosylase. The resulting abasic (AP) site is repaired by reinsertion of a single nucleotide by the sequential action of 5'. AP endonuclease, DNA deoxyribophosphodiesterase, DNA polymerase and DNA ligase. Many prokaryotic organisms can gro w somewhere in the range 60 to I 130C. Since BER involves DNA chain breakage as part of the repair process, possibly resulting in thermal disruption of the DNA chain, we asked the question whether such organisms exhibit the same BER as organisms living at moderate temperatures. Surprisingly, we discovered that two thermophilic archaeons did not show the same BER as demonstrated in Escherichia coli and eukaryotic cells. Instead of first being joined to the 3'-end by a common DNA polymerase reaction, the in coming deoxynucleotide i s first joined to the 5-end before the DNA strand is sealed. Our object is to characterise the enzymatic mechanisms of this novel BER, and to clone and over-express genes/purify enzymes involved in the process.