Abstract
cDNAs for the Xenopus laevis homologue of the endo/exonuclease FEN-1 (DNase IV) have been cloned using a polymerase chain reaction strategy. Products were obtained from two nonallelic Xenopus genes (xFEN-1a and xFEN-1b) that differ from each other by 4.5% in amino acid sequence. Both are 80% identical to mammalian FEN-1 proteins and 55% identical to the yeast homologues. When expressed in Escherichia coli, the Xenopus enzymes showed flap endonuclease activity, a unique feature of this class of nucleases. In addition, expression from the Xenopus cDNAs complemented the temperature and methyl methanesulfonate sensitivity of a yeast rad27 deletion, which eliminates the endogenous FEN-1 gene product. Antiserum raised against xFEN-1 was used to show that the protein accumulates during the middle and late stages of oogenesis, in parallel with other DNA metabolic activities, and that it is localized to the oocyte nucleus. Flap endonuclease activity was demonstrated in oocyte nuclear extracts, and this was inhibited by the anti-xFEN-1 antiserum. The antiserum did not inhibit the major oocyte 5' --> 3' exonuclease activity. DNA synthesis in oocyte extracts was blocked by the antiserum, and the nature of this inhibition suggests that xFEN-1 may be part of a large complex of replication factors. Chromatographic evidence was obtained for the existence of a complex that forms during DNA synthesis and includes proliferating cell nuclear antigen in addition to xFEN-1. These observations support a critical role for xFEN-1 in DNA replication, but indicate that another enzyme must be responsible for the exonuclease function required for homologous recombination in Xenopus oocytes.
Highlights
The nuclease that is commonly designated FEN-1 was initially isolated and partially characterized in 1969 by Lindahl et al [6], who called it DNase IV
X. laevis has a pseudotetraploid genome [42], and as is typical for genes isolated from this organism, we recovered two classes of cDNAs
These differ from each other in amino acid sequence by 4.5% and in DNA sequence by 9% in the coding region and more extensively in the 5Ј- and 3Ј-untranslated regions. xFEN-1a was represented more abundantly than xFEN-1b both in the oocyte PCR products and in the clones from the embryo library, but clearly both were expressed as RNA in these stages
Summary
The nuclease that is commonly designated FEN-1 was initially isolated and partially characterized in 1969 by Lindahl et al [6], who called it DNase IV. Eliminating RAD27 function in yeast renders the cells sensitive to DNA-methylating agents, like methyl methanesulfonate [17,18,19] This suggests a role for FEN-1 in base excision repair, a notion that is supported by biochemical findings with reconstituted systems (26 –29). Inhibition by oligonucleotides annealed to the distal end of the branch implies that the enzyme loads on the free 5Ј-end and travels to the branch point before cleavage [14, 30] These activities are consistent with functions in the trimming of the 5Ј-ends of Okazaki fragments during replication and in the removal of damaged sites during repair. Kim et al [29] isolated xFEN-1 and demonstrated its capabilities in base excision repair
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