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...
There has been a dramatic increase in the detection of lung nodules, many of which are preneoplasia atypical adenomatous hyperplasia (AAH), adenocarcinoma in situ (AIS), minimally invasive adenocarcinoma (MIA) or invasive adenocarcinoma (ADC). The molecular landscape and the evolutionary trajectory of lung preneoplasia have not been well defined. Here, we perform multi-region exome sequencing of 116 resected lung nodules including AAH (n = 22), AIS (n = 27), MIA (n = 54) and synchronous ADC (n = 13). Comparing AAH to AIS, MIA and ADC, we observe progressive genomic evolution at the single nucleotide level and demarcated evolution at the chromosomal level supporting the early lung carcinogenesis model from AAH to AIS, MIA and ADC. Subclonal analyses reveal a higher proportion of clonal mutations in AIS/MIA/ADC than AAH suggesting neoplastic transformation of lung preneoplasia is predominantly associated with a selective sweep of unfit subclones. Analysis of multifocal pulmonary nodules from the same patients reveal evidence of convergent evolution.
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