The GAN exonuclease, or the flap endonuclease Fen1 and RNase HII are necessary for viability of Thermococcus kodakarensis.
Brett W. Burkhart, Lubomira Cubonova, Margaret R. Heider, John N. Reeve, Thomas J. Santangelo, Zvi Kelman
Many aspects of and factors required for DNA replication are conserved across all three Domains of life but there are some significant differences surrounding lagging strand synthesis. In Archaea a 5' to 3' exonuclease, related to both bacterial RecJ and eukaryotic Cdc45, that associates with the replisome, specifically through interactions with GINS, was identified and designated GAN (for GINS- associated nuclease). Despite the presence of a well-characterized flap endonuclease (Fen1), it was hypothesized that GAN might participate in primer removal during Okazaki fragment maturation and, as a Cdc45 homologue, GAN might also be a structural component of an archaeal CMG (Cdc45, MCM, GINS) replication complex. We demonstrate here that, individually, either Fen1 or GAN can be deleted with no discernable effects on viability and growth. However, deletion of both Fen1 and GAN was not possible consistent with both enzymes catalyzing the same step in primer removal from Okazaki fragments in vivo. RNase HII has also been proposed to participate in primer processing during Okazaki fragment maturation. Strains with both Fen1 and RNase HII deleted grew well. GAN activity is therefore sufficient for viability in the absence of both RNase HII and Fen1 but, it was not possible to construct a strain with both RNase HII and GAN deleted. Fen1 alone is therefore insufficient for viability in the absence of both RNase HII and GAN. The ability to delete GAN demonstrates that GAN is not required for activation or stability of the archaeal MCM replicative helicase.
, Cubonova, L.
, Heider, M.
, Reeve, J.
, Santangelo, T.
and Kelman, Z.
The GAN exonuclease, or the flap endonuclease Fen1 and RNase HII are necessary for viability of Thermococcus kodakarensis., Journal of Bacteriology, [online], https://doi.org/10.1128/JB.00141-17
(Accessed May 29, 2023)