MCM8IP activates the MCM8-9 helicase to promote DNA synthesis and homologous recombination upon DNA damage

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regulator of MCM8-9-dependent DNA synthesis during DNA recombination and replication.


Statistics
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Data
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Life sciences study design
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Sample size
No statistical method was used to predetermine sample size. The samples sizes selected for quantitative studies are based on previous studies in the field with similar methodologies (e.g.,

Replication
Cell-based assays (cell survival, homologous recombination assays, MCM8IP localization) and biochemical reactions (EMSA and helicase assays) were reproduced in at least 3 independent biological replicates. Western blotting, immunoprecipitation and pulldown assays are representative of at least 2 independent experiments each. The DNA fiber and RAD51 foci experiments are representative of at least 2 biological replicates. All attempts at replication were successful with quantitative data analyzed for statistical significance.
Randomization There was no randomization in this study as the nature of our experiments did not require them.

Blinding
Data from survival assays, HR repair assays, RAD51 and RPA foci assays, and EMSA and helicase assays were collected and analyzed digitally with consistent settings, and therefore did not require blinding. Blinding was not feasible for western blotting and interaction studies and would unlikely influence outcome. DNA fiber assays and MCM8IP-FLAG localization assays (foci and UV-damaged tract formation) were not blinded as they were stained with commercially available and widely-used antibodies that yielded robust signals for scoring by eye. Additionally, they were conducted appropriately with respect to experimental setup (i.e., untreated controls), data collection (i.e., microscopic fields chosen at random and imaged with the same settings) and data analysis (i.e., scoring under the same image settings). Following our analyses, the phenotypes were apparent and we are therefore confident that blinding would not have yielded meaningfully different outcomes.

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Validation
The antibodies used in this study are available commercially with specificity and application data available online. Applications in our study for each antibody are listed in the previous section and in the manuscript. In particular, the specificities of C17orf53 (both anti-rabbit and anti-mouse), MCM8 and MCM9 antibodies were each validated with some combination of sgRNA-mediated nature research | reporting summary October 2018 deletion, siRNA-induced depletion, and/or cDNA (wildtype and mutant) overexpression studies in our cell lines by western blotting and/or immunoprecipitation, many of which are presented in the manuscript. Furthermore, specificity of the MCM9 antibody was previously reported (Park et al, PMID: 23401855

Authentication
Cell lines were not independently authenticated.

Mycoplasma contamination
Cell lines were not independently tested for mycoplasma contamination.
Commonly misidentified lines (See ICLAC register) No commonly misidentified cell lines were used in this study (ICLAC,v10).

Flow Cytometry Plots
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Methodology
Sample preparation U2OS DR-GFP, U2OS DR-GFP-derived MCM8IP-KO clone and U2OS 35S (SCR/RFP) cells were seeded in 6-well tissue culture plates so as to achieve 60-70% confluency the following day. Cells were then transfected with 2.5 μg of an I-SceI expression vector or pEGFP-N3. In parallel experiments, control cells (non-targeting or empty vector-complemented KO cells) were transfected with 2.5 μg of pCAGGS (empty vector control) to determine GFP/RFP-positivity. Two days after transfection, cells were trypsinized, washed once in PBS, and subjected to flow cytometry.
HEK293T-BFP+ cells were seeded in 24-well tissue culture plates so as to achieve 50-70% confluency the following day. Cells were then co-transfected with 250 ng of pX330 (SpCas9-expressing plasmid containing a BFP-targeting sgRNA) and 500 ng of plasmid containing an HDR donor sequence. Three days after transfection, cells were trypsinized, washed once in PBS, and subjected to flow cytometry.

Instrument
BD LSRII (DR-GFP assay) or BD LSRFortessa (SCR/RFP assay and BFP conversion assay) Software BD FACSDiva Software 8.0 Cell population abundance No post-sort fractions were collected.

Gating strategy
For U2OS DR-GFP, U2OS DR-GFP-derived MCM8IP-KO clone and U2OS 35S (SCR/RFP) cells, FSC/SSC parameters were initially used to gate the majority of the population to the exclusion of cell debris. From that gated population, FITC-A/PE-A parameters were then used to determine GFP-and/or RFP-positivity (HR repair events) in the DR-GFP and SCR-RFP assays. Discerning positivity from negativity was determined by initially positioning gates in pCAGGS-transfected (negative control) and I-SceItransfected (positive control) non-targeting or empty vector-complemented KO cells. Once established, the same gates were then applied to MCM8IP sgRNA-expressing or MCM8IP cDNA-complemented KO cells. A similar gating strategy for determining GFP-positivity in our transfection efficiency controls (pEGFP-N3-transfected cells) was used but with SSC-A/FITC-A parameters.
For HEK239T-BFP+ cells, FSC/SSC parameters were initially used to gate the majority of the population to the exclusion of cell debris. From that gated population, SSC-A/FITC-A parameters were then used to determine GFP-positivity (HR repair events) in the BFP conversion assay. Discerning positivity from negativity was determined by initially positioning gates in pX330-BFP sgRNAtransfected (negative control) control cells and cells co-transfected with px330-BFP sgRNA and an HDR donor (positive control). Once established, the same gates were then applied to MCM8IP sgRNA-expressing cells.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.