C. elegans orthologs MUT-7/CeWRN-1 of Werner syndrome protein regulate neuronal plasticity

Caenorhabditis elegans expresses human Werner syndrome protein (WRN) orthologs as two distinct proteins: MUT-7, with a 3′−5′ exonuclease domain, and CeWRN-1, with helicase domains. How these domains cooperate remains unclear. Here, we demonstrate the different contributions of MUT-7 and CeWRN-1 to 22G small interfering RNA (siRNA) synthesis and the plasticity of neuronal signaling. MUT-7 acts specifically in the cytoplasm to promote siRNA biogenesis and in the nucleus to associate with CeWRN-1. The import of siRNA by the nuclear Argonaute NRDE-3 promotes the loading of the heterochromatin-binding protein HP1 homolog HPL-2 onto specific loci. This heterochromatin complex represses the gene expression of the guanylyl cyclase ODR-1 to direct olfactory plasticity in C. elegans. Our findings suggest that the exonuclease and helicase domains of human WRN may act in concert to promote RNA-dependent loading into a heterochromatin complex, and the failure of this entire process reduces plasticity in postmitotic neurons.

Checkpoint Defects Elicit a WRNIP1-Mediated Response to Counteract R-Loop-Associated Genomic Instability

Conflicts between replication and transcription are a common source of genomic instability, a characteristic of almost all human cancers. Aberrant R-loops can cause a block to replication fork progression. A growing number of factors are involved in the resolution of these harmful structures and many perhaps are still unknown. Here, we reveal that the Werner interacting protein 1 (WRNIP1)-mediated response is implicated in counteracting aberrant R-loop accumulation. Using human cellular models with compromised Ataxia-Telangiectasia and Rad3-Related (ATR)-dependent checkpoint activation, we show that WRNIP1 is stabilized in chromatin and is needed for maintaining genome integrity by mediating the Ataxia Telangiectasia Mutated (ATM)-dependent phosphorylation of Checkpoint kinase 1 (CHK1). Furthermore, we demonstrated that loss of Werner Syndrome protein (WRN) or ATR signaling leads to formation of R-loop-dependent parental ssDNA upon mild replication stress, which is covered by Radiorestistance protein 51 (RAD51). We prove that Werner helicase-interacting protein 1 (WRNIP1) chromatin retention is also required to stabilize the association of RAD51 with ssDNA in proximity of R-loops. Therefore, in these pathological contexts, ATM inhibition or WRNIP1 abrogation is accompanied by increased levels of genomic instability. Overall, our findings suggest a novel function for WRNIP1 in preventing R-loop-driven genome instability, providing new clues to understand the way replication–transcription conflicts are handled.

Investigation of the core binding regions of human Werner syndrome and Fanconi anemia group J helicases on replication protein A

Werner syndrome protein (WRN) and Fanconi anemia group J protein (FANCJ) are human DNA helicases that contribute to genome maintenance. They interact with replication protein A (RPA), and these interactions dramatically enhance the unwinding activities of both helicases. Even though the interplay between these helicases and RPA is particularly important in the chemoresistance pathway of cancer cells, the precise binding regions, interfaces, and properties have not yet been characterized. Here we present systematic NMR analyses and fluorescence polarization anisotropy assays of both helicase-RPA interactions for defining core binding regions and binding affinities. Our results showed that two acidic repeats of human WRN bind to RPA70N and RPA70A. For FANCJ, the acidic-rich sequence in the C-terminal domain is the binding region for RPA70N. Our results suggest that each helicase interaction has unique features, although they both fit an acidic peptide into a basic cleft for RPA binding. Our findings shed light on the protein interactions involved in overcoming the DNA-damaging agents employed in the treatment of cancer and thus potentially provide insight into enhancing the efficacy of cancer therapy.

Werner Syndrome Protein and DNA Replication

Werner Syndrome (WS) is an autosomal recessive disorder characterized by the premature development of aging features. Individuals with WS also have a greater predisposition to rare cancers that are mesenchymal in origin. Werner Syndrome Protein (WRN), the protein mutated in WS, is unique among RecQ family proteins in that it possesses exonuclease and 3′ to 5′ helicase activities. WRN forms dynamic sub-complexes with different factors involved in DNA replication, recombination and repair. WRN binding partners either facilitate its DNA metabolic activities or utilize it to execute their specific functions. Furthermore, WRN is phosphorylated by multiple kinases, including Ataxia telangiectasia mutated, Ataxia telangiectasia and Rad3 related, c-Abl, Cyclin-dependent kinase 1 and DNA-dependent protein kinase catalytic subunit, in response to genotoxic stress. These post-translational modifications are critical for WRN to function properly in DNA repair, replication and recombination. Accumulating evidence suggests that WRN plays a crucial role in one or more genome stability maintenance pathways, through which it suppresses cancer and premature aging. Among its many functions, WRN helps in replication fork progression, facilitates the repair of stalled replication forks and DNA double-strand breaks associated with replication forks, and blocks nuclease-mediated excessive processing of replication forks. In this review, we specifically focus on human WRN’s contribution to replication fork processing for maintaining genome stability and suppressing premature aging. Understanding WRN’s molecular role in timely and faithful DNA replication will further advance our understanding of the pathophysiology of WS.

Residues in the RecQ C-terminal Domain of the Human Werner Syndrome Helicase Are Involved in Unwinding G-quadruplex DNA

The structural and biophysical properties typically associated with G-quadruplex (G4) structures render them a significant block for DNA replication, which must be overcome for cell division to occur. The Werner syndrome protein (WRN) is a RecQ family helicase that has been implicated in the efficient processing of G4 DNA structures. The aim of this study was to identify the residues of WRN involved in the binding and ATPase-driven unwinding of G4 DNA. Using a c-Myc G4 DNA model sequence and recombinant WRN, we have determined that the RecQ-C-terminal (RQC) domain of WRN imparts a 2-fold preference for binding to G4 DNA relative to non-G4 DNA substrates. NMR studies identified residues involved specifically in interactions with G4 DNA. Three of the amino acids in the WRN RQC domain that exhibited the largest G4-specific changes in NMR signal were then mutated alone or in combination. Mutating individual residues implicated in G4 binding had a modest effect on WRN binding to DNA, decreasing the preference for G4 substrates by ∼25%. Mutating two G4-interacting residues (T1024G and T1086G) abrogated preferential binding of WRN to G4 DNA. Very modest decreases in G4 DNA-stimulated ATPase activity were observed for the mutant enzymes. Most strikingly, G4 unwinding by WRN was inhibited ∼50% for all three point mutants and >90% for the WRN double mutant (T1024G/T1086G) relative to normal B-form dsDNA substrates. Our work has helped to identify residues in the WRN RQC domain that are involved specifically in the interaction with G4 DNA.

The Werner Syndrome Helicase Coordinates Sequential Strand Displacement and FEN1-Mediated Flap Cleavage during Polymerase δ Elongation

ABSTRACT The Werner syndrome protein (WRN) suppresses the loss of telomeres replicated by lagging-strand synthesis by a yet to be defined mechanism. Here, we show that whereas either WRN or the Bloom syndrome helicase (BLM) stimulates DNA polymerase δ progression across telomeric G-rich repeats, only WRN promotes sequential strand displacement synthesis and FEN1 cleavage, a critical step in Okazaki fragment maturation, at these sequences. Helicase activity, as well as the conserved winged-helix (WH) motif and the helicase and RNase D C-terminal (HRDC) domain play important but distinct roles in this process. Remarkably, WRN also influences the formation of FEN1 cleavage products during strand displacement on a nontelomeric substrate, suggesting that WRN recruitment and cooperative interaction with FEN1 during lagging-strand synthesis may serve to regulate sequential strand displacement and flap cleavage at other genomic sites. These findings define a biochemical context for the physiological role of WRN in maintaining genetic stability.