G-quadruplex structures inhibit telomerase activity and must be disrupted for telomere elongation during S phase. It has been suggested that the replication protein A (RPA) could unwind and maintain single-stranded DNA in a state amenable to the binding of telomeric components. We show here that under near-physiological in vitro conditions, human RPA is able to bind and unfold G-quadruplex structures formed from a 21mer human telomeric sequence. Analyses by native gel electrophoresis, cross-linking and fluorescence resonance energy transfer indicate the formation of both 1:1 and 2:1 complexes in which G-quadruplexes are unfolded. In addition, quadruplex opening by hRPA is much faster than observed with the complementary DNA, demonstrating that this protein efficiently unfolds G-quartets. A two-step mechanism accounting for the binding of hRPA to G-quadruplexes is proposed. These data point to the involvement of hRPA in regulation of telomere maintenance.

Replication Protein A is a single-stranded (ss) DNA-binding protein that is highly conserved in eukaryotes and plays essential roles in many aspects of nucleic acid metabolism, including replication, recombination, DNA repair and telomere maintenance. It is a heterotrimeric complex consisting of three subunits: RPA1, RPA2 and RPA3. It possesses four DNA-binding domains (DBD), DBD-A, DBD-B and DBD-C in RPA1 and DBD-D in RPA2, and it binds ssDNA via a multistep pathway. Unlike the RPA1 and RPA2 subunits, no ssDNA-RPA3 interaction has as yet been observed although RPA3 contains a structural motif found in the other DBDs. We show here using 4-thiothymine residues as photoaffinity probe that RPA3 interacts directly with ssDNA on the 3′-side on a 31 nt ssDNA.

535 Background: DFS has been widely adopted as a clinically meaningful primary endpoint in EBC trials in place of OS, which needs extensive follow-up to observe sufficient events and fails to assess survival benefit early. However, a rigorous validation of DFS as a surrogate endpoint for OS is required for each given setting. The objective of this analysis was to evaluate DFS as surrogate for OS in the adjuvant treatment of HR+/HER2− EBC. Methods: A systematic literature review (SLR) was conducted using biomedical literature databases (ie, Embase, PubMed, and Medline [from database inception to January 2023]) and key conferences to identify published studies in EBC. The eligibility criteria for the SLR were RCTs that included ≥80% of adult pts with HR+/HER2− EBC and assessing different treatment options (ie, cyclin-dependent kinase 4/6 inhibitors [CDK4/6i], endocrine therapies [ET], and chemotherapies [CT]). Studies were considered for correlation analysis if hazard ratios (HRs) were reported for both DFS and OS. The analysis excluded observational studies and trials with immature OS data. Spearman rank correlation (rs) and weighted linear regression analyses (measured by coefficient of determination [R2]) were performed to evaluate the correlation between HRs for OS and DFS. Considering heterogeneity, multiple scenario analyses were conducted. Surrogate threshold effect (STE) was also estimated; STE is defined as the maximum value of the HR for DFS that needs to be observed in a trial to ensure the possibility of concluding that a significant effect on OS occurred. Correlation was also assessed using pt-level data from the FACE trial (letrozole vs anastrozole in postmenopausal pts with HR+/HER2− EBC) to validate findings of the trial-level analysis. Results: A total of 14 RCTs (N = 31,668) were included (CDK4/6, 2; ET, 7; CT, 5). Trial-level analysis showed that rs between OS and DFS was 0.81 (95% CI, 0.56-0.94), which when weighted by variance was 0.81. Regression analysis showed that 84% of the variability in OS was explained by DFS (R2 = 0.84). A meta-regression model estimated an STE of 0.82 for HR of DFS, which can be leveraged to validate the surrogacy based on the DFS estimated from a future trial. Analysis of pt-level data from the FACE trial demonstrated a correlation between DFS and OS (rs = 0.75). Additionally, correlation coefficients estimated by the Pearson method and iterative imputation were 0.83 and 0.89, respectively. Conclusions: Both trial-level and pt-level analyses demonstrate a positive correlation between DFS and OS. These results support the use of DFS as a reliable surrogate endpoint for OS in HR+/HER2− EBC trials to give an early access to innovative therapies.