Intracellular dynamics of archaeal FANCM homologue Hef in response to halted DNA replication.
Hef is an archaeal member of the DNA repair endonuclease XPF (XPF)/Crossover junction endonuclease MUS81 (MUS81)/Fanconi anemia, complementation group M (FANCM) protein family that in eukaryotes participates in the restart of stalled DNA replication forks. To investigate the physiological roles of Hef in maintaining genome stability in living archaeal cells, we studied the localization of Hef-green fluorescent protein fusions by fluorescence microscopy. Our studies revealed that Haloferax volcanii Hef proteins formed specific localization foci under regular growth conditions, the number of which specifically increased in response to replication arrest. Purification of the full-length Hef protein from its native host revealed that it forms a stable homodimer in solution, with a peculiar elongated configuration. Altogether our data indicate that the shape of Hef, significant physicochemical constraints and/or interactions with DNA limit the apparent cytosolic diffusion of halophilic DNA replication/repair complexes, and demonstrate that Hef proteins are dynamically recruited to archaeal eukaryotic-like chromatin to counteract DNA replication stress. We suggest that the evolutionary conserved function of Hef/FANCM proteins is to enhance replication fork stability by directly interacting with collapsed replication forks.
Laboratoire d'Optique et Biosciences, Ecole Polytechnique, CNRS UMR7645-INSERM U696, 91128 Palaiseau Cedex, France and Section of Molecular Cytology, Swammerdam Institute for Life Sciences, van Leeuwenhoek Centre for Advanced Microscopy, University of Amsterdam, Science Park 904, 1098 XH Amsterdam, The Netherlands.
Roxane
R
Lestini
Sergey P
SP
Laptenok
Joëlle
J
Kühn
Mark A
MA
Hink
Marie-Claire
MC
Schanne-Klein
Ursula
U
Liebl
Hannu
H
Myllykallio
D016590:Aphidicolin / Q000494:pharmacology; D019843:Archaeal Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D048429:Cell Size / Q000187:drug effects; D004249:DNA Damage; D004265:DNA Helicases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D051856:Fanconi Anemia Complementation Group Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000032:analysis; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D019622:Haloferax volcanii / Q000166:cytology / Q000378:metabolism; D045565:Holliday Junction Resolvases / Q000502:physiology; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000032:analysis
D019843:Archaeal Proteins; D051856:Fanconi Anemia Complementation Group Proteins; D005456:Fluorescent Dyes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D016590:Aphidicolin; D045565:Holliday Junction Resolvases; D004265:DNA Helicases