Cdk1 Restrains NHEJ through Phosphorylation of XRCC4-like Factor Xlf1

Summary Eukaryotic cells use two principal mechanisms for repairing DNA double-strand breaks (DSBs): homologous recombination (HR) and nonhomologous end-joining (NHEJ). DSB repair pathway choice is strongly regulated during the cell cycle. Cyclin-dependent kinase 1 (Cdk1) activates HR by phosphorylation of key recombination factors. However, a mechanism for regulating the NHEJ pathway has not been established. Here, we report that Xlf1, a fission yeast XLF ortholog, is a key regulator of NHEJ activity in the cell cycle. We show that Cdk1 phosphorylates residues in the C terminus of Xlf1 over the course of the cell cycle. Mutation of these residues leads to the loss of Cdk1 phosphorylation, resulting in elevated levels of NHEJ repair in vivo. Together, these data establish that Xlf1 phosphorylation by Cdc2Cdk1 provides a molecular mechanism for downregulation of NHEJ in fission yeast and indicates that XLF is a key regulator of end-joining processes in eukaryotic organisms.


Experimental Procedures Genetic and Cell Studies
Media and standard genetic techniques were as described previously (Moreno et al., 1991).
Spot tests and spore survival assays were carried out as in (Hentges et al., 2006) xlf1 phosphorylation mutant strains were created by integrating DNA fragments containing T180A.S192A and T180E.S192E at the xlf1 locus using a cre-lox method described in (Watson et al., 2008). nmt41, nmt1 and nmt41-GFP strains were derived from this by integrating the corresponding cassettes described in . Nat deletion strains were constructed as in . For the taz1d assays, strains were created by integrating a kanMX cassette into the relevant background at the taz1 locus while assays was carried out as described previously (Reis et al., 2012).
To determine the formation of Rad52 foci after IR, nmt1-xlf1 rad52-GFP and nmt1-xlf1.AA rad52-GFP cells were grown to log-phase in YNB without thiamine. A population of early G2 cells was isolated using centrifugation through a lactose gradient. Cells were treated with 50Gy gamma irradiation, attached to a concanavalin A-coated chamber. The presence of Rad52 foci was monitored over 4 hours.

DSB Plasmid Repair Assays
The plasmid religation assay using leucine selection (fig 2.a) was carried out as described previously (Manolis et al., 2001). The plasmid relegation assay using hygromycin selection (fig 2.b) was carried out in the same manner except that plasmid pRL1 bearing a hphMX marker was used, and that plasmid transformation was followed by a 2 hour incubation step before hygromycin selection was applied by plating cells on hygromcyin-containing media. The plasmid repair assay measuring HR and NHEJ in parallel ( fig. 3.d) used plasmids pRL1 (hygromycin) for end-joining and pJK148 (leu1 marker) for chromosomal integration at leu1. 2x10 8 mid-log phase cells were washed and incubated in 4ml DTT buffer for 15min at 30°C. Cells were washed twice with 2ml 1M sorbitol, resuspended in a total volume of 100µl by adding 70µl 1M sorbitol and then divided in two. For the NHEJ electroporations, 200ng uncut pAL19 and 600ng EcoRV and PvuII digested pRL1 plasmid.
NHEJ efficiency was calculated by dividing the number of hygromycin resistant colonies by the number of leucine positive colonies. HR efficiency was calculated by dividing leu+ colonies by the number of hygromycin resistant colonies. Values were normalised to wt = 100% for both repair pathways.

Protein methods
To separate and detect phosphorylated Xlf1 using the Phos-tag, samples were separated by SDS-PAGE on 12% gels containing 25μM Phos-tag (AAL-107 Wako) and 50μM MnCl 2 .
Prior to transfer onto PVDF, gels were incubated for 10min in transfer buffer with 1mM EDTA, then buffer without EDTA, followed by standard Western blotting. GFP-Xlf1 was detected using anti-GFP antibody from Invitrogen (1:2500 dilution).
For the experiment with cdc10.M17 ts cells, cultures were grown to log-phase in EMM2 without thiamine at 25°C, blocked by growth at 36.5°C for one generation time, and released by switching temperature to 25°C. Whole cell lysates were prepared using the TCA method (Watson et al., 2008) and analysed by Western blotting with Phos-tag as described above.
Chk1 phosphorylation was analysed using lysates prepared using the TCA method, and detection of Chk1-HA using monoclonal anti-HA antibody (diluted 1:2000; Santa Cruz Biotechnology).
Recombinant Xlf1 protein with the mutations T180A, S192A, or T180A.S192A was prepared by site-directed mutagenesis of xlf1 on pET28a, followed by expression and purification as described previously (Hentges et al., 2006).