Structure and function of a novel endonuclease acting on branched DNA substrates

International audienceBranched DNA structures that occur during DNA repair and recombination must be efficiently processed by structure-specific endonucleases in order to avoid cell death. In the present paper, we summarize our screen for new interaction partners for the archaeal replication clamp that led to the functional characterization of a novel endonuclease family, dubbed NucS. Structural analyses of Pyrococcus abyssi NucS revealed an unexpected binding site for ssDNA (single-stranded DNA) that directs, together with the replication clamp, the nuclease activity of this protein towards ssDNA-dsDNA (double-stranded DNA) junctions. Our studies suggest that understanding the detailed architecture and dynamic behaviour of the NucS (nuclease specific for ssDNA)-PCNA (proliferating-cell nuclear antigen) complex with DNA will be crucial for identification of its physiologically relevant activities. ©The Authors Journal compilation ©2011 Biochemical Society

Intracellular dynamics of archaeal FANCM homologue Hef in response to halted DNA replication.

International audienceHef 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

NucS-DNA flap association at 23°C.

<p>a) Total number of detected interactions as a function of time (green squares) together with a linear fit (red dashed line <i>N</i> = 0.13<i>t</i>) yielding a constant effective on-rate. b) <i>k_on</i> measured for different substrates at 150 mM NaCl either for wild-type NucS (WT) or a binding-deficient mutant (R70A). 3′-flaps (green), 5′-flaps (blue), 5′flaps with Dig-AntiDig blocked extremity (pink) and dsDNA (red). c) Association rate constant <i>k_on</i> measured for 3′-flaps (green) and 5′-flaps (blue) at different salt concentrations (50 mM and 150 mM NaCl). d) Association rate constant <i>k_on</i> measured for 3′-flaps for different glycerol concentrations (0%, 5% and 20%).</p

Dissociation rates of NucS on DNA flaps and dissociation constants .

<p>Dissociation rates of NucS on DNA flaps and dissociation constants .</p

NucS-DNA dissociation at 23°C.

<p>a) Cumulative proportion of observation time for: (i) photobleaching of NucS-Alexa 488 immobilized on the coverslip in the absence of DNA (blue plain line) fitted by an exponential function (purple dashed line: with <i>t_c</i>  = 2.0 s), (ii) NucS interacting non-specifically with a treated surface (red dashed line) and (iii) NucS interacting with 20-bp long 3′-flaps in 150 mM NaCl buffer (green plain line). The corresponding histogram of raw data for case (iii) is shown in the inset. ***: <i>p<</i>10<i>⁻</i>³. Note that this graph presents the raw residence time distribution and consequently events used to construct the green curve are a mix of photobleaching and dissociation. All the other results presented in this figure (b–d) are dissociation time distributions obtained from the raw distributions as mentioned in the main text. b) NucS cumulative interaction time distribution on 3′-flaps in 150 mM NaCl buffer (green squares) with an exponential fit <i>k_off</i>  = 0.076 s⁻¹(red line). The blue dashed lines are exponential functions using the limit values defined by the standard error (<i>k_off</i>  = 0.069 s⁻¹ and <i>k_off</i>  = 0.082 s⁻¹). c) Interaction time distribution at 50 mM (left) and 150 mM (right) salt for 5′-flaps (blue squares) and 3′-flaps (green circles). d) Left: interaction time distribution for 3′-flaps at different glycerol concentration (blue dashed line: 5%, purple dashed line: 20%) Right: interaction time distribution for 3′-flaps of different length (blue dashed line: 20 bp, red dashed line: 30 bp). On panels c) and d), we present the cumulative distribution of interaction time instead of their distribution to perform comparisons without biases due to the construction of histograms.</p

NucS-DNA flap association rate constants for different salt and glycerol values for the 3′ flap.

<p>NucS-DNA flap association rate constants for different salt and glycerol values for the 3′ flap.</p

Association rate constants at 45°C.

<p>Association rate constants at 45°C.</p

Dissociation rates at 45°C.

<p>Dissociation rates at 45°C.</p