The Fen1 extrahelical 3′-flap pocket is conserved from archaea to human and regulates DNA substrate specificity

AUTOR(ES)

Friedrich-Heineken, Erica

FONTE

Oxford University Press

RESUMO

Fen1 is a key enzyme for the maintenance of genetic stability in archaea and eukaryotes and is classified as a tumor suppressor. Very recent structural data obtained from Archaeoglobus fulgidus Fen1 suggest that an extrahelical 3′-flap pocket is responsible for substrate specificity, by binding to the unpaired 3′-flap and by opening and kinking the DNA. Since the extrahelical 3′-flap pocket in archaeal Fen1 contains seven amino acids that are conserved to a great extent in human Fen1, we have mutated the four conserved or all seven amino acids in the human Fen1 extrahelical 3′-flap pocket to alanine. Our data suggest that the human extrahelical 3′-flap pocket mutants have lost substrate specificity to the double-flap DNA. Moreover, loss of high affinity for the unpaired 3′-flap suggests that the extrahelical 3′-flap pocket is essential for recognition and processing of the ‘physiological’ template. Human PCNA could stimulate the human Fen1 extrahelical 3′-flap pocket mutants but not restore their specificity. Thus the substrate specificity of Fen1 has been functionally conserved over a billion years from archaea to human.

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