Academic literature on the topic 'Flap Endo-exoNuclease; Cell cycle'

ABSTRACT Despite the wealth of information available on the biochemical functions and our recent findings of its roles in genome stability and cancer avoidance of the structure-specific flap endonuclease 1 (FEN1), its cellular compartmentalization and dynamics corresponding to its involvement in various DNA metabolic pathways are not yet elucidated. Several years ago, we demonstrated that FEN1 migrates into the nucleus in response to DNA damage and under certain cell cycle conditions. In the current paper, we found that FEN1 is superaccumulated in the nucleolus and plays a role in the resolution of stalled DNA replication forks formed at the sites of natural replication fork barriers. In response to UV irradiation and upon phosphorylation, FEN1 migrates to nuclear plasma to participate in the resolution of UV cross-links on DNA, most likely employing its concerted action of exonuclease and gap-dependent endonuclease activities. Based on yeast complementation experiments, the mutation of Ser187Asp, mimicking constant phosphorylation, excludes FEN1 from nucleolar accumulation. The replacement of Ser187 by Ala, eliminating the only phosphorylation site, retains FEN1 in nucleoli. Both of the mutations cause UV sensitivity, impair cellular UV damage repair capacity, and decline overall cellular survivorship.

Two processes, DNA replication and DNA damage repair, are key to maintaining genomic fidelity. The Dna2 enzyme lies at the heart of both of these processes, acting in conjunction with flap endonuclease 1 and replication protein A in DNA lagging strand replication and with BLM/Sgs1 and MRN/X in double strand break repair. In vitro, Dna2 helicase and flap endo/exonuclease activities require an unblocked 5′ single-stranded DNA end to unwind or cleave DNA. In this study we characterize a Dna2 nuclease activity that does not require, and in fact can create, 5′ single-stranded DNA ends. Both endonuclease and flap endo/exonuclease are abolished by the Dna2-K677R mutation, implicating the same active site in catalysis. In addition, we define a novel ATP-dependent flap endo/exonuclease activity, which is observed only in the presence of Mn2+. The endonuclease is blocked by ATP and is thus experimentally distinguishable from the flap endo/exonuclease function. Thus, Dna2 activities resemble those of RecB and AddAB nucleases even more closely than previously appreciated. This work has important implications for understanding the mechanism of action of Dna2 in multiprotein complexes, where dissection of enzymatic activities and cofactor requirements of individual components contributing to orderly and precise execution of multistep replication/repair processes depends on detailed characterization of each individual activity.

Exonuclease 1 (Exo1) is a 5′→3′ exonuclease and 5′-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1—a recently identified human SSB that promotes DNA resection during homologous recombination—supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells.

Abstract Background: HPSE is an endo-ß-d-glucuronidase that trims the heparan sulfate (HS) chains of proteoglycans, releasing biologically active fragments of HS. HPSE activity impacts cell signaling, gene expression and promotes extracellular matrix remodeling within the tumor microenvironment; high HPSE expression is associated with enhanced tumor growth, angiogenesis and metastases in several cancer types. As a result of its tumor promoting activities, HPSE is a promising new and unexploited target for anti-cancer therapy. There is a single enzymatically active HPSE in humans and HPSE knockout mice appear to be healthy, thus therapeutic neutralization of HPSE activity would likely have limited negative side effects. In MM preclinical models HPSE was shown to be a master regulator of aggressive tumor behavior. Preclinical evidence also indicates that HPSE promotes chemoresistance suggesting it plays a pivotal role in regulating myeloma response to therapy (Ramani VPC et al., AACR 2014, Abstract nr. 1708). In preclinical studies, bortezomib or melphalan were found to enhance HPSE expression and secretion. High HPSE expressing MM cells were less susceptible to the cytotoxic effects of those drugs. Likewise, a very significant increase in HPSE gene expression following chemotherapy was observed in patient-derived tumor samples, indicating a potential role for HPSE in regulating myeloma response to therapy. Roneparstat (SST0001), a 100% N-acetylated and glycol split heparin, is a potent HPSE inhibitor devoid of any significant anticoagulant activity. In an in vivo model of disseminated myeloma, Roneparstat in combination with either bortezomib or melphalan, significantly decreased both the number of animals with detectable tumor and the tumor burden when compared with animals treated with either of these drugs alone. In addition, studies in animal models of MM indicated that the mechanism of action of Roneparstat was consistent with it having anti-HPSE activity in vivo (reduced angiogenesis and diminished expression of HGF, VEGF and MMP-9 and diminished HPSE induced shedding of syndecan-1, a HS proteoglycan known to be a potent promoter of myeloma growth). Patients and Methods: A First in Man, multicenter, international, phase I clinical study is currently ongoing in advanced heavily pre-treated refractory MM patients (pts) who have exhausted all available anti-MM therapies. Roneparstat is administered subcutaneously, with a starting flat dose defined according to ICH S9 guidelines. A schedule DX5W1,W2 Q28D is being tested. Each cohort plans 3 + 3 pts. A direct fluorescence method (Heparin Red assay) is used in pharmacokinetic studies along with aPTT, used as a surrogate (indirect) measurement of Roneparstat plasma concentration. The pharmacodynamic effect of the drug on the coagulation cascade and any antitumor effect are also evaluated. Results: 15 pts have been enrolled to date. 5 cohorts (doses ranging from 25 to 200 mg/day) have been evaluated, while a 400 mg cohort has just been opened. Five pts have received 1 cycle of therapy, six pts 2 cycles, one 3 cycles, one 5 cycles, one 9 cycles; one patient is currently on treatment, one is not evaluable. Roneparstat administration was found to be safe with only minimal transient side effects. No DLTs and no bleeding complications have been observed. Roneparstat has been well tolerated both systemically and locally. The only side effect observed was minor reactions (redness, bruising) at the injection site (in 6 pts, all grade 1). A decrease > 50% in the serum monoclonal component was observed in one patient, lasting for 6 cycles. Conclusions: Preclinical studies in MM lines and animal models have demonstrated Roneparstat as a potent anti-myeloma compound, particularly when used in combination with other drugs. In the ongoing Phase I escalating dose study (n. pts = 15), Roneparstat administration (at a dose of up to 200 mg/day) was found to be safe with only minimal local side effects. Based on these results, Roneparstat, at a defined dose, in combination with other anti-myeloma agents, will be evaluated in relapsed/resistant MM pts. Disclosures Galli: sigma-tau Research Switzerland SA: Consultancy. Einsele:Novartis: Consultancy, Honoraria, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen/Onyx: Consultancy, Honoraria, Speakers Bureau. Barbieri:sigma-tau Research Switzerland SA, Mendrisio, Switzerland: Employment. Paoletti:sigma-tau Research Switzerland SA, Mendrisio, Switzerland: Employment. Pace:sigma-sau Industrie Farmaceutiche Riunite SpA, Pomezia (RM), Italy: Employment. Sanderson:Sigma-tau Research S.P.A.: Consultancy, Research Funding. Nagler:Novaratis Pharmaceuticals Corporation: Consultancy, Honoraria, Research Funding.