Genome-wide mapping of helicase-generated ssDNA reveals Hrq1 activity at RNA polymerase III-transcribed genes

Technical Summary: Genome-wide mapping of helicase-generated ssDNA reveals Hrq1 activity at RNA polymerase III-transcribed genes

Problem Statement
DNA helicases are essential for maintaining genome integrity by unwinding DNA during replication, repair, recombination, and transcription. However, defining their precise sites of action in vivo remains a significant challenge. Traditional methods like ChIP-seq often fail to capture transient or functional interactions, particularly for helicases that are difficult to purify or possess complex domain structures. Specifically, the human RecQ4 helicase (RECQL4), linked to Rothmund-Thomson syndrome and cancer, is poorly understood due to technical difficulties in studying its large, disordered N-terminal domain and its potential off-target effects on replication initiation. The yeast homolog, Hrq1, serves as a functional model for the helicase core of RECQL4, yet its genome-wide activity profile was previously unknown.

Methodology
The authors developed a novel sequencing-based strategy to map helicase activity genome-wide by coupling helicases to the single-stranded DNA (ssDNA)-specific enzyme, Activation-Induced Cytidine Deaminase (AID).

• AID Fusion Strategy: The core premise is that helicase-mediated DNA unwinding exposes ssDNA, which serves as a substrate for AID. AID deaminates cytosines to uracils within WRC motifs on ssDNA. Upon DNA replication and sequencing, these deaminations appear as strand-specific C-to-T (or G-to-A) mutations, creating a "mutational footprint" of helicase engagement.
• Experimental Systems:
  1. Direct Fusion: A multicopy plasmid expressing Hrq1-AID (or catalytically dead Hrq1-KA-AID) was induced in hrq1Δ yeast strains.
  2. Inducible Dimerization: To avoid overexpression artifacts, an endogenous HRQ1-FRB strain was used with a galactose-inducible AID-FKBP12 construct. Rapamycin-induced dimerization recruited AID to endogenous Hrq1.
  3. Validation: The method was validated using the well-characterized PIF1-family helicase Rrm3.
• Controls and Conditions: Experiments were conducted in ung1Δ strains to prevent base excision repair of uracils. Controls included empty vectors, AID-only, and helicase-dead mutants. Conditions included normal growth, mild replication stress (hydroxyurea), and various genetic backgrounds (e.g., pif1-m2 to remove nuclear Pif1).
• Sequencing and Analysis: Whole-genome sequencing (Illumina) was performed on a custom W303 reference genome. Variants were called with high stringency, and enrichment was analyzed against genomic features (tRNA genes, RNAPII genes, replication origins/ACS, G-quadruplexes) using hypergeometric tests.

Key Results

1. Hrq1 Enrichment at RNAPIII Genes: The genome-wide map revealed that Hrq1 activity is highly enriched at RNA Polymerase III (RNAPIII)-transcribed genes, specifically tRNA genes. In two independent replicates, Hrq1-dependent deaminations showed a 27- to 62-fold enrichment at tRNA loci compared to expectation.
2. Template Strand Specificity: Deaminations occurred predominantly on the transcriptional template strand of RNAPIII genes. This bias was observed in both overexpression and endogenous dimerization systems. The signal was significantly reduced in the helicase-dead Hrq1-KA-AID mutant, confirming that active DNA unwinding drives the association.
3. Universality of Association: Hrq1 associated with nearly all tRNA genes regardless of transcription levels, replication orientation (head-on vs. co-directional), proximity to transposable elements, or intron status.
4. Validation with Rrm3: Applying the same approach to Rrm3 recovered its known enrichment at a subset of highly transcribed, head-on tRNA genes, particularly in the absence of nuclear Pif1 or under replication stress. This confirmed the method's ability to accurately map helicase activity in vivo.
5. Lack of Transcriptional Impact: Despite the strong association with RNAPIII genes, neither deletion nor overexpression of Hrq1 altered pre-tRNA abundance or RNAPIII recycling under the tested conditions (exponential growth, temperature stress, or Tfc1 depletion).
6. Other Genomic Features: Modest enrichment was observed at replication origins (ACS) and G-quadruplexes, but these signals largely disappeared when tRNA-proximal regions were excluded, suggesting they are secondary to the primary tRNA association.

Significance and Claims
The paper establishes AID-mediated mutational footprinting as a general, high-resolution approach for mapping helicase activity in vivo, overcoming limitations of traditional chromatin-based methods.

• Novel Discovery: The study provides the first genome-wide map of Hrq1 activity, revealing an unexpected, widespread association with RNAPIII-transcribed genes that was not previously characterized.
• Functional Insight: While Hrq1 is physically associated with the template strand of active RNAPIII genes, the authors conclude that it is not required for steady-state transcription or recycling under unperturbed conditions. The authors hypothesize that Hrq1 may play a surveillance role in resolving transcription-associated damage or R-loops at these loci, a function that would not necessarily alter pre-tRNA levels in the absence of stress.
• Model for RECQL4: By characterizing Hrq1 in a system free of the confounding Sld2-like replication initiation domain found in human RECQL4, the study clarifies the conserved helicase functions of the RecQ4 family.
• Methodological Utility: The successful application to Rrm3 validates the technique as a robust tool for investigating the in vivo activity of other ssDNA-binding proteins and helicases.

The authors note limitations, including low deamination frequencies requiring deep sequencing and the inability to fully decouple transcription-derived ssDNA from helicase-derived ssDNA, but emphasize that the method successfully identifies functional helicase engagement sites with near-nucleotide resolution.