Centromeres consist of DNA repeats in many eukaryotes. Non-allelic homologous recombination (HR) between them can result in gross chromosomal rearrangements (GCRs). In fission yeast, Rad51 suppresses isochromosome formation that occurs between inverted repeats in the centromere. However, how the HR enzyme prevents homology-mediated GCRs remains unclear. Here, we provide evidence that Rad51 with the aid of the Swi/Snf-type motor protein Rad54 promotes non-crossover recombination between centromere repeats to prevent isochromosome formation. Mutations in Rad51 and Rad54 epistatically increased the rates of isochromosome formation and chromosome loss. In sharp contrast, these mutations decreased gene conversion between inverted repeats in the centromere. Remarkably, analysis of recombinant DNAs revealed that rad51 and rad54 increase the proportion of crossovers. In the absence of Rad51, deletion of the structure-specific endonuclease Mus81 decreased both crossovers and isochromosomes, while the cdc27/pol32-D1 mutation, which impairs break-induced replication, did not. We propose that Rad51 and Rad54 promote non-crossover recombination between centromere repeats on the same chromatid, thereby suppressing crossover between non-allelic repeats on sister chromatids that leads to chromosomal rearrangements. Furthermore, we found that Rad51 and Rad54 are required for gene silencing in centromeres, suggesting that HR also plays a role in the structure and function of centromeres.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are associated with increased risk for developing Parkinson's disease (PD). Previously, we found that LRRK2 G2019S mutation carriers have increased mitochondrial DNA (mtDNA) damage and after zinc finger nuclease-mediated gene mutation correction, mtDNA damage was no longer detectable. While the mtDNA damage phenotype can be unambiguously attributed to the LRRK2 G2019S mutation, the underlying mechanism(s) is unknown. Here, we examine the role of LRRK2 kinase function in LRRK2 G2019S-mediated mtDNA damage, using both genetic and pharmacological approaches in cultured neurons and PD patient-derived cells. Expression of LRRK2 G2019S induced mtDNA damage in primary rat midbrain neurons, but not in cortical neuronal cultures. In contrast, the expression of LRRK2 wild type or LRRK2 D1994A mutant (kinase dead) had no effect on mtDNA damage in either midbrain or cortical neuronal cultures. In addition, human LRRK2 G2019S patient-derived lymphoblastoid cell lines (LCL) demonstrated increased mtDNA damage relative to age-matched controls. Importantly, treatment of LRRK2 G2019S expressing midbrain neurons or patient-derived LRRK2 G2019S LCLs with the LRRK2 kinase inhibitor GNE-7915, either prevented or restored mtDNA damage to control levels. These findings support the hypothesis that LRRK2 G2019S-induced mtDNA damage is LRRK2 kinase activity dependent, uncovering a novel pathological role for this kinase. Blocking or reversing mtDNA damage via LRRK2 kinase inhibition or other therapeutic approaches may be useful to slow PD-associated pathology.

Homologous recombination between repetitive sequences can lead to gross chromosomal rearrangements (GCRs). At fission yeast centromeres, Rad51-dependent conservative recombination predominantly occurs between inverted repeats, thereby suppressing formation of isochromosomes whose arms are mirror images. However, it is unclear how GCRs occur in the absence of Rad51 and how GCRs are prevented at centromeres. Here, we show that homology-mediated GCRs occur through Rad52-dependent single-strand annealing (SSA). The rad52-R45K mutation, which impairs SSA activity of Rad52 protein, dramatically reduces isochromosome formation in rad51 deletion cells. A ring-like complex Msh2-Msh3 and a structure-specific endonuclease Mus81 function in the Rad52-dependent GCR pathway. Remarkably, mutations in replication fork components, including DNA polymerase α and Swi1/Tof1/Timeless, change the balance between Rad51-dependent recombination and Rad52-dependent SSA at centromeres, increasing Rad52-dependent SSA that forms isochromosomes. Our results uncover a role of DNA replication machinery in the recombination pathway choice that prevents Rad52-dependent GCRs at centromeres.

Zafar F., Seidler S.B., Kronenberg A., Schild D. and Wiese C. Homologous recombination contributes to the repair of DNA double-strand breaks induced by high-energy iron ions. Radiat. Res.To test the contribution of homologous recombinational repair (HRR) in repairing DNA damaged sites induced by high-energy iron ions, we used: 1) HRR-deficient rodent cells carrying a deletion in the RAD51D gene and 2) syngeneic human cells impaired for HRR by RAD51D or RAD51 knockdown using RNA interference. We show that in response to iron ions, HRR contributes to cell survival in rodent cells, and that HRR-deficiency abrogates RAD51 foci formation. Complementation of the HRR defect by human RAD51D rescues both enhanced cytotoxicity and RAD51 foci formation. For human cells irradiated with iron ions, cell survival is decreased, and, in p53 mutant cells, the levels of mutagenesis are increased when HRR is impaired. Human cells synchronized in S phase exhibit more pronounced resistance to iron ions as compared with cells in G1 phase, and this increase in radioresistance is diminished by RAD51 knockdown. These results implicate a role for RAD51-mediated DNA repair (i.e. HRR) in removing a fraction of clustered lesions induced by charged particle irradiation. Our results are the first to directly show the requirement for an intact HRR pathway in human cells in ensuring DNA repair and cell survival in response to high-energy high LET radiation.3

The “Iowa kindred,” a large Iowan family with autosomal-dominant Parkinson’s disease, has been followed clinically since the 1920s at the Mayo Clinic. In 2003, the genetic cause was determined to be a 1.7 Mb triplication of the alpha-synuclein genomic locus. Affected individuals present with an early-onset, severe parkinsonism-dementia syndrome. Here, we present a descendant of the Iowa kindred with novel, disease-associated non-motor findings of reduced heart rate variability, complete anosmia, and a rare skin condition called colloid milium. At autopsy, key neuropathological findings were compatible with diffuse Lewy body disease. Using high-resolution comparative genomic hybridization (CGH) array analysis to fine-map the genomic breakpoints, we observed two independent recombination events of the SNCA locus that resulted in a genomic triplication of twelve genes, including SNCA, and the disruption of two genes, HERC6 and CCSER1, at the genomic breakpoints. In conclusion, we provide further evidence that the mere two-fold overexpression of alpha-synuclein leads to a fulminant alpha-synucleinopathy with rapid progression and severe clinical and neuropathological features.