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.

We report the genome-wide mapping of ORC1 binding sites in mammals, by chromatin immunoprecipitation and parallel sequencing (ChIP-seq). ORC1 binding sites in HeLa cells were validated as active DNA replication origins (ORIs) using Repli-seq, a method that allows identification of ORI-containing regions by parallel sequencing of temporally ordered replicating DNA. ORC1 sites were universally associated with transcription start sites (TSSs) of coding or noncoding RNAs (ncRNAs). Transcription levels at the ORC1 sites directly correlated with replication timing, suggesting the existence of two classes of ORIs: those associated with moderate/high transcription levels ($1 RNA copy/cell), firing in early S and mapping to the TSSs of coding RNAs; and those associated with low transcription levels (<1 RNA copy/cell), firing throughout the entire S and mapping to TSSs of ncRNAs. These findings are compatible with a scenario whereby TSS expression levels influence the efficiency of ORC1 recruitment at G 1 and the probability of firing during S.[Supplemental material is available for this article.] DNA replication is a highly orchestrated process that ensures fidelity of genomes during duplications, as well as their adaptation to variations in cell division, DNA damage, and, in metazoa, chromatin changes associated with development and differentiation. It initiates from multiple chromosomal loci, called replication origins (ORIs), which are selected in the G 1 phase of the cell cycle by sequential recruitment of the origin recognition complex (ORC), CDC6, CDT1, and the MCM complex (the pre-replicative complex; pre-RC). Selected pre-RCs are then sequentially activated during the S phase, following a tight temporally ordered program (Mechali 2010).In Saccharomyces cerevisiae, ORIs contain a 12-bp consensus for ORC binding (Bell and Stillman 1992). Genome-wide analyses of ORIs by chromatin immunoprecipitation and parallel sequencing (ChIP-seq) using anti-ORC or -MCM antibodies showed that this consensus is essential but not sufficient for origin activity and identified other features that influence selection and replication timing, including transcription and/or chromatin structure (Eaton et al. 2010). In metazoa, instead, pre-RC does not exhibit sequence specificity, and the number of potential ORIs is considerably larger, following a process of selection that differs according to cell type, functional status, or stress conditions (Mechali 2010).These further levels of complexity allow DNA replication to adapt to the unique expression patterns of individual cell types. Little is known, however, about the regulation of ORI selection and replication timing in metazoa.

Cellular and biochemical studies support a role for all five human RecQ helicases in DNA replication; however, their specific functions during this process are unclear. Here we investigate the in vivo association of the five human RecQ helicases with three well-characterized human replication origins. We show that only RECQ1 (also called RECQL or RECQL1) and RECQ4 (also called RECQL4) associate with replication origins in a cell cycle-regulated fashion in unperturbed cells. RECQ4 is recruited to origins at late G 1 , after ORC and MCM complex assembly, while RECQ1 and additional RECQ4 are loaded at origins at the onset of S phase, when licensed origins begin firing. Both proteins are lost from origins after DNA replication initiation, indicating either disassembly or tracking with the newly formed replisome. Nascent-origin DNA synthesis and the frequency of origin firing are reduced after RECQ1 depletion and, to a greater extent, after RECQ4 depletion. Depletion of RECQ1, though not that of RECQ4, also suppresses replication fork rates in otherwise unperturbed cells. These results indicate that RECQ1 and RECQ4 are integral components of the human replication complex and play distinct roles in DNA replication initiation and replication fork progression in vivo.The RecQ helicases are a family of DNA-unwinding enzymes essential for the maintenance of genome integrity in all kingdoms of life. Five RecQ enzymes have been found in human cells: RECQ1 (also called RECQL or RECQL1), BLM (RECQ2 or RECQL3), WRN (RECQ3 or RECQL2), RECQ4 (RECQL4), and RECQ5 (RECQL5) (3, 7). Here we refer to these helicases as RECQ1, RECQ4, and RECQ5, without the "L" that is present in the official gene names. Mutations in the BLM, WRN, and RECQ4 genes are linked to Bloom syndrome (BS), Werner syndrome (WS), and the subset of RothmundThomson syndrome (RTS) patients at high risk of developing osteosarcomas, respectively (19,31,71). RECQ4 mutations have also been associated with RAPADILINO and BallerGerold syndrome (56, 61). Although these disorders are all associated with inherent genomic instability and cancer predisposition, they show distinct clinical features, suggesting that BLM, WRN, and RECQ4 are involved in different aspects of DNA metabolism. However, the molecular events underlying the pathogenesis of BS, WS, and RTS remain obscure. Mutations in the remaining two human RecQ helicase genes, RECQ1 and RECQ5, have not as yet been identified as causes of either genomic instability or heritable cancer predisposition disorders.Several lines of evidence suggest that RecQ helicases play an important role in DNA replication control (3, 10). In particular, RecQ helicases are thought to facilitate replication by preserving the integrity of stalled replication forks and by remodeling or repairing damaged or collapsed forks to allow the resumption of replication. Consistent with these ideas, several investigators have shown that primary fibroblasts from BS, WS, and RTS patients and RecQ5-deficient mouse embryonic fibroblasts all show differential...

RecQ helicases have attracted considerable interest in recent years due to their role in the suppression of genome instability and human diseases. These atypical helicases exert their function by resolving a number of highly specific DNA structures. The crystal structure of a truncated catalytic core of the human RECQ1 helicase (RECQ149–616) shows a prominent β-hairpin, with an aromatic residue (Y564) at the tip, located in the C-terminal winged-helix domain. Here, we show that the β-hairpin is required for the DNA unwinding and Holliday junction (HJ) resolution activity of full-length RECQ1, confirming that it represents an important determinant for the distinct substrate specificity of the five human RecQ helicases. In addition, we found that the β-hairpin is required for dimer formation in RECQ149–616 and tetramer formation in full-length RECQ1. We confirmed the presence of stable RECQ149–616 dimers in solution and demonstrated that dimer formation favours DNA unwinding; even though RECQ1 monomers are still active. Tetramers are instead necessary for more specialized activities such as HJ resolution and strand annealing. Interestingly, two independent protein–protein contacts are required for tetramer formation, one involves the β-hairpin and the other the N-terminus of RECQ1, suggesting a non-hierarchical mechanism of tetramer assembly.