A mRad51-GFP antimorphic allele affects homologous recombination and DNA damage sensitivity.

Accurate DNA double-strand break repair through homologous recombination is essential for preserving genome integrity. Disruption of the gene encoding RAD51, the protein that catalyzes DNA strand exchange during homologous recombination, results in lethality of mammalian cells. Proteins required for homologous recombination, also play an important role during DNA replication. To explore the role of RAD51 in DNA replication and DSB repair, we used a knock-in strategy to express a carboxy-terminal fusion of green fluorescent protein to mouse RAD51 (mRAD51-GFP) in mouse embryonic stem cells. Compared to wild-type cells, heterozygous mRad51(+/wt-GFP) embryonic stem cells showed increased sensitivity to DNA damage induced by ionizing radiation and mitomycin C. Moreover, gene targeting was found to be severely impaired in mRad51(+/wt-GFP) embryonic stem cells. Furthermore, we found that mRAD51-GFP foci were not stably associated with chromatin. From these experiments we conclude that this mRad51-GFP allele is an antimorphic allele. When this allele is present in a heterozygous condition over wild-type mRad51, embryonic stem cells are proficient in DNA replication but display defects in homologous recombination and DNA damage repair.

Resolution of telomere associations by TRF1 cleavage in mouse embryonic stem cells.

Telomere associations have been observed during key cellular processes such as mitosis, meiosis, and carcinogenesis and must be resolved before cell division to prevent genome instability. Here we establish that telomeric repeat-binding factor 1 (TRF1), a core component of the telomere protein complex, is a mediator of telomere associations in mammalian cells. Using live-cell imaging, we show that expression of TRF1 or yellow fluorescent protein (YFP)-TRF1 fusion protein above endogenous levels prevents proper telomere resolution during mitosis. TRF1 overexpression results in telomere anaphase bridges and aggregates containing TRF1 protein and telomeric DNA. Site-specific protein cleavage of YFP-TRF1 by tobacco etch virus protease resolves telomere aggregates, indicating that telomere associations are mediated by TRF1. This study provides novel insight into the formation and resolution of telomere associations.

The mammalian proteins MMS19, MIP18, and ANT2 are involved in cytoplasmic iron-sulfur cluster protein assembly.

Iron-sulfur (Fe-S) clusters are essential cofactors of proteins with a wide range of biological functions. A dedicated cytosolic Fe-S cluster assembly (CIA) system is required to assemble Fe-S clusters into cytosolic and nuclear proteins. Here, we show that the mammalian nucleotide excision repair protein homolog MMS19 can simultaneously bind probable cytosolic iron-sulfur protein assembly protein CIAO1 and Fe-S proteins, confirming that MMS19 is a central protein of the CIA machinery that brings Fe-S cluster donor proteins and the receiving apoproteins into proximity. In addition, we show that mitotic spindle-associated MMXD complex subunit MIP18 also interacts with both CIAO1 and Fe-S proteins. Specifically, it binds the Fe-S cluster coordinating regions in Fe-S proteins. Furthermore, we show that ADP/ATP translocase 2 (ANT2) interacts with Fe-S apoproteins and MMS19 in the CIA complex but not with the individual proteins. Together, these results elucidate the composition and interactions within the late CIA complex.

RTEL1 contributes to DNA replication and repair and telomere maintenance.

Telomere maintenance and DNA repair are important processes that protect the genome against instability. mRtel1, an essential helicase, is a dominant factor setting telomere length in mice. In addition, mRtel1 is involved in DNA double-strand break repair. The role of mRtel1 in telomere maintenance and genome stability is poorly understood. Therefore we used mRtel1-deficient mouse embryonic stem cells to examine the function of mRtel1 in replication, DNA repair, recombination, and telomere maintenance. mRtel1-deficient mouse embryonic stem cells showed sensitivity to a range of DNA-damaging agents, highlighting its role in replication and genome maintenance. Deletion of mRtel1 increased the frequency of sister chromatid exchange events and suppressed gene replacement, demonstrating the involvement of the protein in homologous recombination. mRtel1 localized transiently at telomeres and is needed for efficient telomere replication. Of interest, in the absence of mRtel1, telomeres in embryonic stem cells appeared relatively stable in length, suggesting that mRtel1 is required to allow extension by telomerase. We propose that mRtel1 is a key protein for DNA replication, recombination, and repair and efficient elongation of telomeres by telomerase.

RTEL1: an essential helicase for telomere maintenance and the regulation of homologous recombination.

Telomere maintenance and DNA repair are crucial processes that protect the genome against instability. RTEL1, an essential iron-sulfur cluster-containing helicase, is a dominant factor that controls telomere length in mice and is required for telomere integrity. In addition, RTEL1 promotes synthesis-dependent strand annealing to direct DNA double-strand breaks into non-crossover outcomes during mitotic repair and in meiosis. Here, we review the role of RTEL1 in telomere maintenance and homologous recombination and discuss models linking RTEL1's enzymatic activity to its function in telomere maintenance and DNA repair.

The microtubule plus-end-tracking protein CLIP-170 associates with the spermatid manchette and is essential for spermatogenesis.

CLIP-170 is a microtubule "plus-end-tracking protein" implicated in the control of microtubule dynamics, dynactin localization, and the linking of endosomes to microtubules. To investigate the function of mouse CLIP-170, we generated CLIP-170 knockout and GFP-CLIP-170 knock-in alleles. Residual CLIP-170 is detected in lungs and embryos of homozygous CLIP-170 knockout mice, but not in other tissues and cell types, indicating that we have generated a hypomorphic mutant. Homozygous CLIP-170 knockout mice are viable and appear normal. However, male knockout mice are subfertile and produce sperm with abnormal heads. Using the knock-in mice, we followed GFP-CLIP-170 expression and behavior in dissected, live testis tubules. We detect plus-end-tracking GFP-CLIP-170 in spermatogonia. As spermatogenesis proceeds, GFP-CLIP-170 expression increases and the fusion protein strongly marks syncytia of differentiated spermatogonia and early prophase spermatocytes. Subsequently GFP-CLIP-170 levels drop, but during spermiogenesis (post-meiotic development), GFP-CLIP-170 accumulates again and is present on spermatid manchettes and centrosomes. Bleaching studies show that, as spermatogenesis progresses, GFP-CLIP-170 converts from a mobile plus-end-tracking protein to a relatively immobile protein. We propose that CLIP-170 has a structural function in the male germline, in particular in spermatid differentiation and sperm head shaping.

Ubiquitin ligase Rad18Sc localizes to the XY body and to other chromosomal regions that are unpaired and transcriptionally silenced during male meiotic prophase.

In replicative damage bypass (RDB) in yeast, the ubiquitin-conjugating enzyme RAD6 interacts with the ubiquitin ligase RAD18. In the mouse, these enzymes are represented by two homologs of RAD6, HR6a and HR6b, and one homolog of RAD18, Rad18Sc. Expression of these genes and the encoded proteins is ubiquitous, but there is relatively high expression in the testis. We have studied the subcellular localization by immunostaining Rad18Sc and other RDB proteins in mouse primary spermatocytes passing through meiotic prophase in spermatogenesis. The highest Rad18Sc protein level is found at pachytene and diplotene, and the protein localizes mainly to the XY body, a subnuclear region that contains the transcriptionally inactivated X and Y chromosomes. In spermatocytes that carry translocations for chromosomes 1 and 13, Rad18Sc protein concentrates on translocation bivalents that are not fully synapsed. The partly synapsed bivalents are often localized in the vicinity of the XY body, and show a very low level of RNA polymerase II, indicating that the chromatin is in a silent configuration similar to transcriptional silencing of the XY body. Thus, Rad18Sc localizes to unsynapsed and silenced chromosome segments during the male meiotic prophase. All known functions of RAD18 in yeast are related to RDB. However, in contrast to Rad18Sc, expression of UBC13 and poleta, known to be involved in subsequent steps of RDB, appears to be diminished in the XY body and regions containing the unpaired translocation bivalents. Taken together, these observations suggest that the observed subnuclear localization of Rad18Sc may involve a function outside the context of RDB. This function is probably related to a mechanism that signals the presence of unsynapsed chromosomal regions and subsequently leads to transcriptional silencing of these regions during male meiotic prophase.