SUMMARY Topoisomerase I (TOP1) inhibitors are an important class of anticancer drugs. The cytotoxicity of TOP1 inhibitors can be modulated by replication fork reversal, in a process that requires PARP activity. Whether regressed forks can efficiently restart and the factors required to restart fork progression after fork reversal are still unknown. Here we combined biochemical and electron microscopy approaches with single-molecule DNA fiber analysis, to identify a key role for human RECQ1 helicase in replication fork restart after TOP1 inhibition, not shared by other human RecQ proteins. We show that the poly(ADPribosyl)ation activity of PARP1 stabilizes forks in their regressed state by limiting their restart by RECQ1. These studies provide new mechanistic insights into the roles of RECQ1 and PARP in DNA replication and offer molecular perspectives to potentiate chemotherapeutic regimens based on TOP1 inhibition.

Following prolonged genotoxic stress, DNA2 and WRN functionally interact to degrade reversed replication forks and promote replication restart, thereby preventing aberrant processing of unresolved replication intermediates

RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration.DNA helicases | RecQ | genome stability | Holliday junction | fork reversal R ecQ helicases are a family of ATP-dependent motor proteins that play central roles in maintaining genome stability. Defects in three of the five human RecQ homologs give rise to distinct genetic disorders associated with genomic instability, cancer predisposition, and premature aging (1-5). The unique clinical features of these disorders support the notion that the different RecQ helicases have nonoverlapping functions, but the molecular basis for their different enzymatic activities remains unclear. RecQ helicases catalyze ATP-dependent DNA unwinding in the 3′-5′ direction. Additionally, members of this helicase family have been shown to tackle an unparalleled breath of noncanonical DNA structures, such as fork DNA, G-quadruplexes, D-loops, and Holliday junction (HJ) structures (6-8). However, our understanding of the physical mechanisms by which RecQ helicases recognize and process their physiological substrates remains remarkably limited.RECQ1 is the shortest of the human RecQ-family helicases, comprising the bipartite ATPase domain common to all superfamily 2 (SF2) helicases, the RecQ-specific C-terminal domain (RQC), and short extensions on the N and C termini. We recently discovered a specific function of RECQ1 in branch migration and restart of reversed DNA replication forks upon DNA topoisomerase I inhibition that is not shared by other human RecQ helicases, such as Werner (WRN) or Bloom (BLM) syndrome proteins (9). On the other hand, BLM is the sole human RecQ helicase member specifically able to resolve double-HJ junction structures in conjunction with DNA topoisomerase III alpha and the RMI1 and RMI2 accessory proteins (10-12). These findings lead us to hypothesize that the sp...

Replicative hexameric helicases are thought to unwind duplex DNA by steric exclusion (SE) where one DNA strand is encircled by the hexamer and the other is excluded from the central channel. However, interactions with the excluded strand on the exterior surface of hexameric helicases have also been shown to be important for DNA unwinding, giving rise to the steric exclusion and wrapping (SEW) model. For example, the archaeal minichromosome maintenance (MCM) helicase has been shown to unwind DNA via a SEW mode to enhance unwinding efficiency. Using single-molecule FRET, we now show that the analogous () DnaB helicase also interacts specifically with the excluded DNA strand during unwinding. Mutation of several conserved and positively charged residues on the exterior surface of DnaB resulted in increased interaction dynamics and states compared with wild type. Surprisingly, these mutations also increased the DNA unwinding rate, suggesting that electrostatic contacts with the excluded strand act as a regulator for unwinding activity. In support of this, experiments neutralizing the charge of the excluded strand with a morpholino substrate instead of DNA also dramatically increased the unwinding rate. Of note, although the stability of the excluded strand was nearly identical forDnaB and MCM, these enzymes are from different superfamilies and unwind DNA with opposite polarities. These results support the SEW model of unwinding forDnaB that expands on the existing SE model of hexameric helicase unwinding to include contributions from the excluded strand to regulate the DNA unwinding rate.

The MCM8/9 complex is implicated in aiding fork progression and facilitating homologous recombination (HR) in response to several DNA damage agents. MCM9 itself is an outlier within the MCM family containing a long C-terminal extension (CTE) comprising 42% of the total length, but with no known functional components and high predicted disorder. In this report, we identify and characterize two unique motifs within the primarily unstructured CTE that are required for localization of MCM8/9 to sites of mitomycin C (MMC)-induced DNA damage. First, an unconventional “bipartite-like” nuclear localization (NLS) motif consisting of two positively charged amino acid stretches separated by a long intervening sequence is required for the nuclear import of both MCM8 and MCM9. Second, a variant of the BRC motif (BRCv) similar to that found in other HR helicases is necessary for localization to sites of MMC damage. The MCM9-BRCv directly interacts with and recruits RAD51 downstream to MMC-induced damage to aid in DNA repair. Patient lymphocytes devoid of functional MCM9 and discrete MCM9 knockout cells have a significantly impaired ability to form RAD51 foci after MMC treatment. Therefore, the disordered CTE in MCM9 is functionally important in promoting MCM8/9 activity and in recruiting downstream interactors; thus, requiring full-length MCM9 for proper DNA repair.