To start DNA replication, the Origin Recognition Complex (ORC) and Cdc6 load a Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase loading intermediate. Here we report a 3.9Å structure of the OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHD) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an α-helix in Orc4 and positively charged loops in Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by a Mcm5 loop that embraces Mcm2, but the N-tier ring Mcm2-Mcm5 interface remains open. This structure suggests loading mechanics of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.

SummaryThe Fanconi anemia (FA) core complex promotes the tolerance/repair of DNA damage at stalled replication forks by catalyzing the monoubiquitination of FANCD2 and FANCI. Intriguingly, the core complex component FANCM also catalyzes branch migration of model Holliday junctions and replication forks in vitro. Here we have characterized the ortholog of FANCM in fission yeast Fml1 in order to understand the physiological significance of this activity. We show that Fml1 has at least two roles in homologous recombination—it promotes Rad51-dependent gene conversion at stalled/blocked replication forks and limits crossing over during mitotic double-strand break repair. In vitro Fml1 catalyzes both replication fork reversal and D loop disruption, indicating possible mechanisms by which it can fulfill its pro- and antirecombinogenic roles.

The formation of healthy gametes depends on programmed DNA double strand breaks (DSBs), which are each repaired as a crossover (CO) or non-crossover (NCO) from a homologous template. Although most of these DSBs are repaired without giving COs, little is known about the genetic requirements of NCO-specific recombination. We show that Fml1, the Fanconi anemia complementation group M (FANCM)-ortholog of Schizosaccharomyces pombe, directs the formation of NCOs during meiosis in competition with the Mus81-dependent pro-CO pathway. We also define the Rad51/Dmc1-mediator Swi5-Sfr1 as a major determinant in biasing the recombination process in favour of Mus81, to ensure the appropriate amount of COs to guide meiotic chromosome segregation. The conservation of these proteins from yeast to Humans suggests that this interplay may be a general feature of meiotic recombination. KeywordsHomologous recombination; Meiosis; Fml1; Mus81; Schizosaccharomyces pombe Faithful chromosome segregation during meiosis depends on the establishment of chiasmata through recombinational repair of programmed DNA double-strand breaks (DSBs) to produce crossovers (COs) between homologous chromosomes (homologs). However, in most cases only a minority of the DSBs are earmarked to form COs, and therefore the majority have to be repaired by using either the homolog without CO formation or the sister chromatid (1).In order to identify helicase activities involved in non-crossover (NCO)-recombination during meiosis in the fission yeast Schizosaccharomyces pombe, we screened for helicases potentially capable of D loop unwinding during synthesis-dependent strand annealing (SDSA), which is thought to be a major pathway of NCO recombination (1). To this end, we used a genetic recombination assay consisting of a meiotic recombination hotspot at the ade6 gene and two flanking scorable markers (Fig. 1A). We hypothesized that at least one of the helicases promoting NCO recombination pathways in mitotic cells would also have a role during meiosis. From our candidate list -fbh1, srs2, rqh1, fml1 and fml2 -only the deletion of fml1 gave the expected increase in CO formation associated with a meiotic gene Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts conversion (GC) event at two different hotspot alleles, ade6-M26 and ade6-3083, and at a non-hotspot allele ade6-M375 (Fig. 1, B and C, and tables S1 to S3) (2-5). Increases in COs were also observed on a different chromosome (Fig. 1D and table S4) and by a physical assay at the mbs1 locus ( fig. S1), indicating that Fml1's role in suppressing CO formation is not restricted to a single locus.In vitro purified Fml1, like its budding yeast ortholog Mph1, unwinds D loops and is therefore suited to promoting SDSA ( Fig. 1E) (6, 7). The fml1-K99R mutant, which encodes protein that retains full DNA binding activity but is unable to unwind D loops ( Fig. 1E and fig. S2), exhibits the same hyper-CO phenotype as the null mutant indicating that Fml1's helicase function is required for NCO...

Combination therapy is increasingly becoming the cornerstone of current day antitumor therapy. Glioblastoma multiforme is an aggressive brain tumor with a dismal median survival post diagnosis and a high rate of disease recurrence. The poor prognosis can be attributed to unique treatment limitations, which include the infiltrative nature of tumor cells, failure of anti-glioma drugs to cross the blood–brain barrier, tumor heterogeneity and the highly metastatic and angiogenic nature of the tumor making cells resistant to chemotherapy. Combination therapy approach is being developed against glioblastoma with new innovative combination drug regimens being tested in preclinical and clinical trials. In this review, we discuss the pathophysiology of glioblastoma, diagnostic markers, therapeutic targeting strategies, current treatment limitations, novel combination therapies in the context of current treatment options and the ongoing clinical trials for glioblastoma therapy.

Fanconi Anemia (FA) is a rare, inherited genomic instability disorder, caused by mutations in genes involved in the repair of interstrand DNA crosslinks (ICLs). The FA signaling network contains a unique nuclear protein complex that mediates the monoubiquitylation of the FANCD2 and FANCI heterodimer, and coordinates activities of the downstream DNA repair pathway including nucleotide excision repair, translesion synthesis, and homologous recombination. FA proteins act at different steps of ICL repair in sensing, recognition and processing of DNA lesions. The multi-protein network is tightly regulated by complex mechanisms, such as ubiquitination, phosphorylation, and degradation signals that are critical for the maintenance of genome integrity and suppressing tumorigenesis. Here, we discuss recent advances in our understanding of how the FA proteins participate in ICL repair and regulation of the FA signaling network that assures the safeguard of the genome. We further discuss the potential application of designing small molecule inhibitors that inhibit the FA pathway and are synthetic lethal with DNA repair enzymes that can be used for cancer therapeutics.