Structural Biology of Genome Maintenance and DNA repair

National Institute Of Environmental Health Sciences

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Abstract

Progress 2024-2025 1) ZATT (Znf451) is a novel vertebrate RNAse Zinc-finger Associated with TDP2 and TOP2 (ZATT, aka ZNF451) is a multifunctional protein participating in the repair of TOP2 mediated DNA damage, DNA replication, and transcription. These nuclear activities have been linked to its intrinsic N-terminal E3 SUMO2/3 ligase activity. ZATT also contains an array of central C2H2 class Zinc finger motifs (Znf), and a C-terminal PIN/NYN family nuclease domain of unknown function. We report that purified ZATT harbors intrinsic ribonuclease (RNase) activity. The broad Mg2+ and Mn2+ dependent ZATT RNase activity that cleaves dsRNA, RNA hairpins and RNA-DNA hybrids in vitro. In the context of embedded ribonucleotides in a DNA scaffold, ZATT displays an RNASEH1-like activity, and efficiently cleaves substrates harboring 4 or more ribonucleotides. A minimal active nucleolytic core encompasses both the Znf repeat and the PIN domain. The PIN core also cleaves RNA targets isolation, but its activity was much less than the Znf-PIN fragments. To define the basis for ZATT catalytic activity, we determined a 2.3 Å resolution crystal structure of ZATT-PIN that unveils an extensive dimeric interface. ZATT exists as a stable dimer in solution as assessed by SEC-MALS, and mutation of the ZATT dimerization interface abrogates RNase activity of full-length (FL) ZATT. Together, results from biochemical and structural analysis point to possible roles for ZATT in the nucleolytic regulation of complex nucleic acid structures like R-loops, which left unresolved are a critical threat to genome integrity. 2) Molecular basis for RNA discrimination by human DNA ligase 1 DNA ligase 1 (LIG1) finalizes DNA replication and repair by catalyzing the joining of DNA nicks. LIG1 is highly specific for DNA-DNA junctions over DNA-RNA junctions, discriminating strongly against a single ribonucleotide at the 5’ side of the nick. This selectivity of LIG1 prevents futile and potentially mutagenic DNA-RNA cleavage and re-ligation cycles during Okazaki fragment maturation or ribonucleotide excision repair of genome embedded rNMPs, but the determinants of LIG1 rNMP discrimination are ill-defined. We report structural and kinetic analysis of LIG1 DNA-RNA complexes showing that LIG1 employs an aromatic steric gate to stabilize the enzyme substrate complex and directly exclude rNMP-containing polynucleotides. Mutation of this RNA gate compromises the adenylyl transfer and nick sealing reactions but decreases the discrimination against an rNMP-containing substrate by ~3600-fold. Our results establish the role of the conserved steric gate in ribonucleotide discrimination by high-fidelity DNA ligases at each step of the ligation reaction, which has parallels to the ribonucleotide discrimination by high-fidelity DNA polymerases. 3) Molecular Architecture and Antiviral Inhibition of the Herpes Simplex Virus-1 Helicase–Primase Complex Greater than 60% of humans are infected with Herpes Simplex Virus (HSV). Infections are chronic, but treatable with antiviral drugs. Pritelivir is a helicase-primase inhibitor (HPI) that blocks HSV-1 and HSV-2 DNA replication through ill-defined mechanisms. Cryo-EM structures of the heterotrimeric HSV-1 UL5–UL52–UL8 complex reveal an integrated catalytic apparatus coordinating DNA unwinding with primer synthesis through close juxtaposition of the UL52 (primase) and UL5 (helicase) active centers. Pritelivir targets the UL5-UL52 interface and wedges between the UL5 RecA1 and RecA2 subdomains to stabilize an opened helicase motor conformation that blocks ATP hydrolysis, thereby precluding helicase translocation, and DNA unwinding. Our results define the molecular operating principles of the HSV-1 helicase-primase assembly and provide insights into HPI resistance.