Cooperation of the N-terminal Helicase and C-terminal endonuclease activities of Archaeal Hef protein in processing stalled replication forks.
Blockage of replication fork progression often occurs during DNA replication, and repairing and restarting stalled replication forks are essential events in all organisms for the maintenance of genome integrity. The repair system employs processing enzymes to restore the stalled fork. In Archaea Hef is a well conserved protein that specifically cleaves nicked, flapped, and fork-structured DNAs. This enzyme contains two distinct domains that are similar to the DEAH helicase family and XPF nuclease superfamily proteins. Analyses of truncated mutant proteins consisting of each domain revealed that the C-terminal nuclease domain independently recognized and incised fork-structured DNA. The N-terminal helicase domain also specifically unwound fork-structured DNA and Holliday junction DNA in the presence of ATP. Moreover, the endonuclease activity of the whole Hef protein was clearly stimulated by ATP hydrolysis catalyzed by the N-terminal domain. These enzymatic properties suggest that Hef efficiently resolves stalled replication forks by two steps, which are branch point transfer to the 5'-end of the nascent lagging strand by the N-terminal helicase followed by template strand incision for leading strand synthesis by the C-terminal endonuclease.
Komori
Kayoko
K
Department of Molecular Biology, Biomolecular Engineering Research Institute, Suita, Osaka 565-0874, Japan.
Hidaka
Masumi
M
Horiuchi
Takashi
T
Fujikane
Ryosuke
R
Shinagawa
Hideo
H
Ishino
Yoshizumi
Y
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D001105:Archaea / Q000378:metabolism*; D019843:Archaeal Proteins / Q000737:chemistry / Q000502:physiology*; D001483:Base Sequence; D015153:Blotting, Western; D002850:Chromatography, Gel; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004720:Endonucleases / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D016680:Genome, Bacterial; D006868:Hydrolysis; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D004720:Endonucleases; D000251:Adenosine Triphosphatases; D004265:DNA Helicases