Biochemical analysis of human PIF1 helicase and functions of its N-terminal domain.
The evolutionary conserved PIF1 DNA helicase family appears to have largely nonoverlapping cellular functions. To better understand the functions of human PIF1, we investigated biochemical properties of this protein. Analysis of single-stranded (ss) DNA-dependent ATPase activity revealed nonstructural ssDNA to greatly stimulate ATPase activity due to a high affinity for PIF1, even though PIF1 preferentially unwinds forked substrates. This suggests that PIF1 needs a ssDNA region for loading and a forked structure for translocation entrance into a double strand region. Deletion analysis demonstrated novel functions of a unique N-terminal portion, named the PIF1 N-terminal (PINT) domain. When the PINT domain was truncated, apparent affinity for ssDNA and unwinding activity were much reduced, even though the maximum velocity of ATPase activity and K(m) value for ATP were not affected. We suggest that the PINT domain contributes to enhancing the interaction with ssDNA through intrinsic binding activity. In addition, we found DNA strand-annealing activity, also residing in the PINT domain. Notably, the unwinding and annealing activities were inhibited by replication protein A. These results suggest that the functions of PIF1 might be restricted with particular situations and DNA structures.
Gu
Yongqing
Y
Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan.
Masuda
Yuji
Y
Kamiya
Kenji
K
D000251:Adenosine Triphosphatases / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D017434:Protein Structure, Tertiary; D051716:Replication Protein A / Q000378:metabolism; D017384:Sequence Deletion
D004277:DNA, Single-Stranded; D051716:Replication Protein A; D004247:DNA; D000251:Adenosine Triphosphatases; D004265:DNA Helicases; C508765:PIF1 protein, human