Abstract
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.
Highlights
The maintenance of genome integrity is a crucial challenge for all proliferating cells
These results indicate that the Hef::green fluorescent protein (GFP) fusion protein is functional in repair of DNA damages caused by mitomycin C (MMC) and aphidicolin, we do not exclude the possibility that Hef proteins might have additional roles in DNA replication or other cellular processes in H. volcanii
To test whether the Hef::GFP localization observed in living cells was specific for aphidicolin, we investigated the localization of Hef::GFP in cells treated with the cross-linking agent MMC [25], the double-strand break-causing agent phleomycin [26] and HU that decreases the size of the deoxyribonucleotide pool [27,28]
Summary
The maintenance of genome integrity is a crucial challenge for all proliferating cells. Mechanistic details for the restart of stalled forks and how this results in genome rearrangements have been described in bacteria [1], yeast [2] and higher eukaryotes [3]. The picture that emerges from numerous studies is that bacterial and eukaryal proteins implicated in homologous recombination play a key role in stabilizing and/or restoring blocked replication forks. In agreement with this notion, inhibiting the elongation phase of DNA replication increases the frequency of replication-coupled recombination as a result of the accumulation of four-branched DNA intermediates that occur during reversal of the blocked replication forks [4]
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