WRNIP1 Protects Reversed DNA Replication Forks from SLX4-Dependent Nucleolytic Cleavage

Abstract

During DNA replication stress, stalled replication forks need to be stabilized to prevent fork collapse and genome instability. The AAA + ATPase WRNIP1 (Werner Helicase Interacting Protein 1) has been implicated in the protection of stalled replication forks from nucleolytic degradation, but the underlying molecular mechanism has remained unclear. Here we show that WRNIP1 exerts its protective function downstream of fork reversal. Unexpectedly though, WRNIP1 is not part of the well-studied BRCA2-dependent branch of fork protection but seems to protect the junction point of reversed replication forks from SLX4-mediated endonucleolytic degradation, possibly by directly binding to reversed replication forks. This function is specific to the shorter, less abundant, and less conserved variant of WRNIP1. Overall, our data suggest that in the absence of BRCA2 and WRNIP1 different DNA substrates are generated at reversed forks but that nascent strand degradation in both cases depends on the activity of exonucleases and structure-specific endonucleases.

Keywords: Biological Sciences; Cell Biology; Molecular Biology.

Graphical abstract

Figure 1

WRNIP1 Protects Reversed Replication Forks in a Different Way Than BRCA2 (A) Scheme of DNA fiber labeling assay. (B) Replication fork degradation analysis upon knock-down of WRNIP1 and codepletion of factors involved in replication fork reversal. (C) Replication fork degradation analysis upon knock-down of WRNIP1 (left) or BRCA2 (right) in the absence or presence of the MRE11 exonuclease inhibitor mirin, the MRE11 endonuclease inhibitor PFM01, or upon codepletion of MRE11. Each dot represents an independent biological replicate. Values and gray bars indicate mean. Statistical analysis: one-way ANOVA with Sidak's correction for multiple comparisons (****, p < 0.0001; *, p < 0.1; ns, not significant). Scatterplots of one representative experiment and western blots showing depletion efficacies can be found in Figure S1. CldU, 5-chloro-2′-deoxyuridine; IdU, 5-iodo-2′-deoxyuridine; HU, hydroxyurea; RNAi, RNA interference; si, siRNA. See also Figure S1 and Tables S1, S3, and S4.

Figure 2

WRNIP1 Protects Reversed Replication Forks from SLX4-Dependent Nucleolytic Cleavage (A) Replication fork degradation analysis upon knock-down of WRNIP1 and codepletion of SLX4 or associated nucleases. Each dot represents an independent biological replicate. Values and gray bars indicate mean. Statistical analysis: one-way ANOVA with Sidak's correction for multiple comparisons (****, p < 0.0001; **, p < 0.01; ns, not significant). Scatterplots of one representative experiment and western blots showing depletion efficacies can be found in Figure S2. (B) EMSA to monitor binding of increasing amounts of WRNIP1 to ssDNA (left), dsDNA (second from left), a replication fork-like substrate (second from right), and a four-way junction substrate (right). See also Figure S2 and Tables S1 and S2–S4.

Figure 3

WRNIP1 Interacts Physically and Functionally with SLX1-SLX4 (A) Endonucleolytic cleavage assay with SLX1-SLX4 in the presence of increasing amounts of WRNIP1. (B) Endonucleolytic cleavage assay with MUS81-EME1 in the presence of increasing amounts of WRNIP1. (C) Coimmunoprecipitation experiment with purified His-tagged SLX1-SLX4 and purified Flag-tagged WRNIP1. (D) Replication fork degradation analysis in WRNIP1-depleted cells, complemented with WRNIP1 variants. Endogenous WRNIP1 was depleted by siRNA targeting the 3′-UTR. Each dot represents an independent biological replicate. Values and gray bars indicate mean. Statistical analysis: one-way ANOVA with Sidak's correction for multiple comparisons (****, p < 0.0001; **, p < 0.01; ns, not significant). Scatterplots of one representative experiment and western blots showing depletion and expression efficacies can be found in Figure S3. 4-WJ, four-way junction; n-ds, nicked duplex DNA; WB, western blot; si, siRNA. See also Figure S3 and Tables S1 and S2–S4.

Figure 4

Fork Resection Requires Endonucleolytic and Exonucleolytic Cleavage Steps (A) Replication fork degradation analysis upon knock-down of WRNIP1 in the absence or presence of the DNA2 inhibitor C5 or upon codepletion of DNA2 or EXO1. (B) Replication fork degradation analysis upon knock-down of BRCA2 and codepletion of SLX4, MUS81, or EME1. Each dot represents an independent biological replicate. Values and gray bars indicate mean. Statistical analysis: one-way ANOVA with Sidak's correction for multiple comparisons (****, p < 0.0001; ***, p < 0.001; ns, not significant). Scatterplots of one representative experiment and western blots showing depletion efficacies can be found in Figure S4. CldU, 5-chloro-2′-deoxyuridine; IdU, 5-iodo-2′-deoxyuridine; HU, hydroxyurea; RNAi, RNA interference; si, siRNA. See also Figure S4 and Tables S1, S3, and S4.

Figure 5

WRNIP1, but Not WRNIP1L, Can Protect Reversed Replication Forks (A) Alignment of WRNIP1's central ATPase domain from a variety of species. Highlighted in bold are the Walker B motif (left) and the arginine finger (right), shaded in gray is the 25 amino acid-long insertion found in WRNIP1L (665 amino acid protein), but not in WRNIP1 (640 amino acid protein). Symbols underneath reflect amino acid conservation, with asterisks marking identical amino acids and colons indicating conservation of chemical character. Hs, Homo sapiens; Mm, Mus musculus; Dd, Dictyostelium discoideum; Sc, Saccharomyces cerevisiae; Sp, Schizosaccharomyces pombe; Ec, Escherichia coli. (B) EMSA to monitor binding of increasing amounts of WRNIP1L to a four-way junction substrate. (C) Endonucleolytic cleavage assay of SLX1-SLX4 in the presence of increasing amounts of WRNIP1L. Dashed line indicates that lanes were left out for clarity. (D) Replication fork degradation analysis upon knock-down of WRNIP1 and complementation with cDNAs expressing the short (WRNIP1) or long (WRNIP1L) isoform. On the right, knock-down of WRNIP1 (short*) or WRNIP1L (long*) using isoform-specific siRNAs. Each dot represents an independent biological replicate. Values and gray bars indicate mean. Statistical analysis: one-way ANOVA with Sidak's correction for multiple comparisons (****, p < 0.0001; ns, not significant). Scatterplots of one representative experiment and western blots showing depletion and expression efficacies can be found in Figure S5. 4-WJ, four-way junction; n-ds, nicked duplex DNA; si, siRNA. See also Figure S5 and Tables S1 and S2–S4.

Figure 6

Model Illustrating the Two Branches of Fork Protection That Are Dependent on BRCA2 and WRNIP1, Respectively See Discussion for details.