The Drosophila Melanogaster RAD54 Homolog, DmRAD54, Is Involved in the Repair of Radiation Damage and Recombination

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRAD54-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X- ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution

WWP2 ubiquitylates RNA polymerase II for DNA-PK-dependent transcription arrest and repair at DNA breaks

DNA double-strand breaks (DSBs) at RNA polymerase II (RNAPII) transcribed genes lead to inhibition of transcription. The DNA-dependent protein kinase (DNA-PK) complex plays a pivotal role in transcription inhibition at DSBs by stimulating proteasome-dependent eviction of RNAPII at these lesions. How DNA-PK triggers RNAPII eviction to inhibit transcription at DSBs remains unclear. Here we show that the HECT E3 ubiquitin ligase WWP2 associates with components of the DNA-PK and RNAPII complexes and is recruited to DSBs at RNAPII transcribed genes. In response to DSBs, WWP2 targets the RNAPII subunit RPB1 for K48-linked ubiquitylation, thereby driving DNA-PK- and proteasome-dependent eviction of RNAPII. The lack of WWP2 or expression of nonubiquitylatable RPB1 abrogates the binding of nonhomologous end joining (NHEJ) factors, including DNA-PK and XRCC4/DNA ligase IV, and impairs DSB repair. These findings suggest that WWP2 operates in a DNA-PK-dependent shutoff circuitry for RNAPII clearance that promotes DSB repair by protecting the NHEJ machinery from collision with the transcription machinery

The NuRD chromatin–remodeling complex regulates signaling and repair of DNA damage

NuRD is recruited to DNA double-strand breaks, where it promotes RNF8/RNF168 histone ubiquitylation and accumulation of DNA repair factors (see also related paper by Larsen et al. in this issue)

Meiosis in Mice without a Synaptonemal Complex

The synaptonemal complex (SC) promotes fusion of the homologous chromosomes (synapsis) and crossover recombination events during meiosis. The SC displays an extensive structural conservation between species; however, a few organisms lack SC and execute meiotic process in a SC-independent manner. To clarify the SC function in mammals, we have generated a mutant mouse strain (Sycp1−/−Sycp3−/−, here called SC-null) in which all known SC proteins have been displaced from meiotic chromosomes. While transmission electron microscopy failed to identify any remnants of the SC in SC-null spermatocytes, neither formation of the cohesion axes nor attachment of the chromosomes to the nuclear membrane was perturbed. Furthermore, the meiotic chromosomes in SC-null meiocytes achieved pre-synaptic pairing, underwent early homologous recombination events and sustained a residual crossover formation. In contrast, in SC-null meiocytes synapsis and MLH1-MLH3-dependent crossovers maturation were abolished, whereas the structural integrity of chromosomes was drastically impaired. The variable consequences that SC inactivation has on the meiotic process in different organisms, together with the absence of SC in some unrelated species, imply that the SC could have originated independently in different taxonomic groups

Genetic Steps of Mammalian Homologous Repair with Distinct Mutagenic Consequences

Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52(−)(/)(−) mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair

Cohesin proteins label the chromosomal axes in SC-null oocytes, but the SC proteins are lost from the chromosomal axes.

<p>(A) SC-null oocytes were stained with antisera recognizing cohesins REC8 (red), RAD21/RAD21L (magenta) and STAG3 (green). (B) SC-null oocytes were labeled with antisera against the central element proteins SYCE1 and SYCE2 (red). Chromosomal axes were identified by labeling of the cohesion protein STAG3 (green). Centromeres, labeled by CREST, are shown in white. Bars, 10 µm.</p

SC-null spermatocytes lack nuclear structures resembling the axial elements or the central region of the SC.

<p>Electron microscopy analysis of nuclei from spermatocytes derived from of wild-type (A, E) and SC-null (B–D) mice. Wild-type pachytene meiocytes show synaptonemal complexes (SC) and normally condensed chromatin (A, E). In SC-null meiocytes, chromatin is less condensed and axial structures are absent (B). The arrow in (B) points to dense regions of centromeric heterochromatin located close to the nuclear envelope. Attachment plates of the nuclear envelope in wild-type and SC-null meiocytes are denoted by arrowheads (C–D). NE, nuclear envelope; XY, XY body.</p

The number of cells used for the statistical analysis of the recombination process.

<p>*number of analyzed cells at the zygotene/early pachytene/late pachytene/diplotene stages.</p><p>**total number of analyzed axial intervals is in parentheses.</p

SC proteins, but not cohesin proteins, are lost from the chromosome axes in SC-null spermatocytes.

<p>(A) SC-null spermatocytes were stained with antisera against the meiosis-specific cohesins REC8 (red) and SMC1β (red) and the cohesin protein STAG3 (green). Centromeres, labeled by CREST, are shown in white. (B) SC-null spermatocytes were labeled with antisera against the axial element protein SYCP2 (red), the central element protein TEX12 (red) or the central element protein SYCE3 (red). The chromosomal axes are identified by labeling of the cohesion protein STAG3 (green). Centromeres, labeled by CREST, are shown in white. Bars, 10 µm.</p