Human premature aging, DNA repair and RecQ helicases

Genomic instability leads to mutations, cellular dysfunction and aberrant phenotypes at the tissue and organism levels. A number of mechanisms have evolved to cope with endogenous or exogenous stress to prevent chromosomal instability and maintain cellular homeostasis. DNA helicases play important roles in the DNA damage response. The RecQ family of DNA helicases is of particular interest since several human RecQ helicases are defective in diseases associated with premature aging and cancer. In this review, we will provide an update on our understanding of the specific roles of human RecQ helicases in the maintenance of genomic stability through their catalytic activities and protein interactions in various pathways of cellular nucleic acid metabolism with an emphasis on DNA replication and repair. We will also discuss the clinical features of the premature aging disorders associated with RecQ helicase deficiencies and how they relate to the molecular defects

New Insights Into DNA Helicases as Druggable Targets for Cancer Therapy

Small molecules that deter the functions of DNA damage response machinery are postulated to be useful for enhancing the DNA damaging effects of chemotherapy or ionizing radiation treatments to combat cancer by impairing the proliferative capacity of rapidly dividing cells that accumulate replicative lesions. Chemically induced or genetic synthetic lethality is a promising area in personalized medicine, but it remains to be optimized. A new target in cancer therapy is DNA unwinding enzymes known as helicases. Helicases play critical roles in all aspects of nucleic acid metabolism. We and others have investigated small molecule targeted inhibition of helicase function by compound screens using biochemical and cell-based approaches. Small molecule-induced trapping of DNA helicases may represent a generalized mechanism exemplified by certain topoisomerase and PARP inhibitors that exert poisonous consequences, especially in rapidly dividing cancer cells. Taking the lead from the broader field of DNA repair inhibitors and new information gleaned from structural and biochemical studies of DNA helicases, we predict that an emerging strategy to identify useful helicase-interacting compounds will be structure-based molecular docking interfaced with a computational approach. Potency, specificity, drug resistance, and bioavailability of helicase inhibitor drugs and targeting such compounds to subcellular compartments where the respective helicases operate must be addressed. Beyond cancer therapy, continued and new developments in this area may lead to the discovery of helicase-interacting compounds that chemically rescue clinically relevant helicase missense mutant proteins or activate the catalytic function of wild-type DNA helicases, which may have novel therapeutic application

Setting the stage for cohesion establishment by the replication fork

Comment on: Rudra S, et al. Cell Cycle 2012; 2114-2

Catalytic Strand Separation by RECQ1 Is Required for RPA-Mediated Response to Replication Stress

SummaryThree (BLM, WRN, and RECQ4) of the five human RecQ helicases are linked to genetic disorders characterized by genomic instability, cancer, and accelerated aging [1]. RECQ1, the first human RecQ helicase discovered [2–4] and the most abundant [5], was recently implicated in breast cancer [6, 7]. RECQ1 is an ATP-dependent DNA-unwinding enzyme (helicase) [8, 9] with roles in replication [10–12] and DNA repair [13–16]. RECQ1 is highly expressed in various tumors and cancer cell lines (for review, see [17]), and its suppression reduces cancer cell proliferation [14], suggesting a target for anti-cancer drugs. RECQ1’s assembly state plays a critical role in modulating its helicase, branch migration (BM), or strand annealing [18, 19]. The crystal structure of truncated RECQ1 [20, 21] resembles that of E. coli RecQ [22] with two RecA-like domains, a RecQ-specific zinc-binding domain and a winged-helix domain, the latter implicated in DNA strand separation and oligomer formation. In addition, a conserved aromatic loop (AL) is important for DNA unwinding by bacterial RecQ [23, 24] and truncated RECQ1 helicases [21]. To better understand the roles of RECQ1, two AL mutants (W227A and F231A) in full-length RECQ1 were characterized biochemically and genetically. The RECQ1 mutants were defective in helicase or BM but retained DNA binding, oligomerization, ATPase, and strand annealing. RECQ1-depleted HeLa cells expressing either AL mutant displayed reduced replication tract length, elevated dormant origin firing, and increased double-strand breaks that could be suppressed by exogenously expressed replication protein A (RPA). Thus, RECQ1 governs RPA’s availability in order to maintain normal replication dynamics, suppress DNA damage, and preserve genome homeostasis

A Partially Functional DNA Helicase II Mutant Defective in Forming Stable Binary Complexes with ATP and DNA: A ROLE FOR HELICASE MOTIF III

To address the functional significance of motif III in Escherichia coli DNA helicase II, the conserved aspartic acid at position 248 was changed to asparagine. UvrDD248N failed to form stable binary complexes with either DNA or ATP. However, UvrDD248N was capable of forming an active ternary complex when both ATP and single-stranded DNA were present. The DNA-stimulated ATPase activity of UvrDD248N was reduced relative to that of wild-type UvrD with no significant change in the apparent Km for ATP. The mutant protein also demonstrated a reduced DNA unwinding activity. The requirement for high concentrations of UvrDD248N to achieve unwinding of long duplex substrates likely reflects the reduced stability of various binary and ternary complexes that must exist in the catalytic cycle of a helicase. The data suggest that motif III may act as an interface between the ATP binding and DNA binding domains of a helicase. The uvrDD248N allele was also characterized in genetic assays. The D248N protein complemented the UV-sensitive phenotype of a uvrD deletion strain to levels nearly equivalent to wild-type helicase II. In contrast, the mutant protein only partially complemented the mutator phenotype. A correlation between the level of genetic complementation and the helicase activity of UvrDD248N is discussed

A Point Mutation in Escherichia coli DNA Helicase II Renders the Enzyme Nonfunctional in Two DNA Repair Pathways: EVIDENCE FOR INITIATION OF UNWINDING FROM A NICKIN VIVO

Biosynthetic errors and DNA damage introduce mismatches and lesions in DNA that can lead to mutations. These abnormalities are susceptible to correction by a number of DNA repair mechanisms, each of which requires a distinct set of proteins. Escherichia coli DNA helicase II has been demonstrated to function in two DNA repair pathways, methyl-directed mismatch repair and UvrABC-mediated nucleotide excision repair. To define further the role of UvrD in DNA repair a site-specific mutant was characterized. The mutation, uvrDQ251E, resides within helicase motif III, a conserved segment of amino acid homology found in a superfamily of prokaryotic and eukaryotic DNA helicases. The UvrD-Q251E protein failed to complement the mutator and ultraviolet light-sensitive phenotypes of a uvrD deletion strain indicating that the mutant protein is inactive in both mismatch repair and excision repair. Biochemical characterization revealed a significant defect in the ability of the mutant enzyme to initiate unwinding at a nick. The elongation phase of the unwinding reaction was nearly normal. Together, the biochemical and genetic data provide evidence that UvrD-Q251E is dysfunctional because the mutant protein fails to initiate unwinding at the nick(s) used to initiate excision and subsequent repair synthesis. These results provide direct evidence to support the notion that helicase II initiates unwinding from a nick in vivo in mismatch repair and excision repair

Sister chromatid cohesion establishment occurs in concert with lagging strand synthesis

Cohesion establishment is central to sister chromatid tethering reactions and requires Ctf7/Eco1-dependent acetylation of the cohesin subunit Smc3. Ctf7/Eco1 is essential during S phase, and a number of replication proteins (RFC complexes, PCNA and the DNA helicase Chl1) all play individual roles in sister chromatid cohesion. While the mechanism of cohesion establishment is largely unknown, a popular model is that Ctf7/Eco1 acetylates cohesins encountered by and located in front of the fork. In turn, acetylation is posited both to allow fork passage past cohesin barriers and convert cohesins to a state competent to capture subsequent production of sister chromatids. Here, we report evidence that challenges this pre-replicative cohesion establishment model. Our genetic and biochemical studies link Ctf7/Eco1 to the Okazaki fragment flap endonuclease, Fen1. We further report genetic and biochemical interactions between Fen1 and the cohesion-associated DNA helicase, Chl1. These results raise a new model wherein cohesin deposition and establishment occur in concert with lagging strand-processing events and in the presence of both sister chromatids

The interaction site of Flap Endonuclease-1 with WRN helicase suggests a coordination of WRN and PCNA

Werner and Bloom syndromes are genetic RecQ helicase disorders characterized by genomic instability. Biochemical and genetic data indicate that an important protein interaction of WRN and Bloom syndrome (BLM) helicases is with the structure-specific nuclease Flap Endonuclease 1 (FEN-1), an enzyme that is implicated in the processing of DNA intermediates that arise during cellular DNA replication, repair and recombination. To acquire a better understanding of the interaction of WRN and BLM with FEN-1, we have mapped the FEN-1 binding site on the two RecQ helicases. Both WRN and BLM bind to the extreme C-terminal 18 amino acid tail of FEN-1 that is adjacent to the PCNA binding site of FEN-1. The importance of the WRN/BLM physical interaction with the FEN-1 C-terminal tail was confirmed by functional interaction studies with catalytically active purified recombinant FEN-1 deletion mutant proteins that lack either the WRN/BLM binding site or the PCNA interaction site. The distinct binding sites of WRN and PCNA and their combined effect on FEN-1 nuclease activity suggest that they may coordinately act with FEN-1. WRN was shown to facilitate FEN-1 binding to its preferred double-flap substrate through its protein interaction with the FEN-1 C-terminal binding site. WRN retained its ability to physically bind and stimulate acetylated FEN-1 cleavage activity to the same extent as unacetylated FEN-1. These studies provide new insights to the interaction of WRN and BLM helicases with FEN-1, and how these interactions might be regulated with the PCNA-FEN-1 interaction during DNA replication and repai