RNase H1 and H2 Are Differentially Regulated to Process RNA-DNA Hybrids.

RNA-DNA hybrids are tightly regulated to ensure genome integrity. The RNase H enzymes RNase H1 and H2 contribute to chromosomal stability through the removal of RNA-DNA hybrids. Loss of RNase H2 function is implicated in human diseases of the nervous system and cancer. To better understand RNA-DNA hybrid dynamics, we focused on elucidating the regulation of the RNase H enzymes themselves. Using yeast as a model system, we demonstrate that RNase H1 and H2 are controlled in different manners. RNase H2 has strict cell cycle requirements, in that it has an essential function in G2/M for both R-loop processing and ribonucleotide excision repair. RNase H1, however, can function independently of the cell cycle to remove R-loops and appears to become activated in response to high R-loop loads. These results provide us with a more complete understanding of how and when RNA-DNA hybrids are acted upon by the RNase H enzymes.

Increased TERRA levels and RNase H sensitivity are conserved hallmarks of post-senescent survivors in budding yeast.

Cancer cells activate telomere maintenance mechanisms (TMMs) to bypass replicative senescence and achieve immortality by either upregulating telomerase or promoting homology-directed repair (HDR) at chromosome ends to maintain telomere length, the latter being referred to as ALT (Alternative Lengthening of Telomeres). In yeast telomerase mutants, the HDR-based repair of telomeres leads to the generation of 'survivors' that escape senescence and divide indefinitely. So far, yeast has proven to provide an accurate model to study the generation and maintenance of telomeres via HDR. Recently, it has been established that up-regulation of the lncRNA, TERRA (telomeric repeat-containing RNA), is a novel hallmark of ALT cells. Moreover, RNA-DNA hybrids are thought to trigger HDR at telomeres in ALT cells to maintain telomere length and function. Here we show that, also in established yeast type II survivors, TERRA levels are increased in an analogous manner to human ALT cells. The elevated TERRA levels are independent of yeast-specific subtelomeric structures, i.e. the presence or absence of Y' repetitive elements. Furthermore, we show that RNase H1 overexpression, which degrades the RNA moiety in RNA-DNA hybrids, impairs the growth of yeast survivors. We suggest that even in terms of TERRA regulation, yeast survivors serve as an accurate model that recapitulates many key features of human ALT cells.

Mte1 interacts with Mph1 and promotes crossover recombination and telomere maintenance.

Mph1 is a member of the conserved FANCM family of DNA motor proteins that play key roles in genome maintenance processes underlying Fanconi anemia, a cancer predisposition syndrome in humans. Here, we identify Mte1 as a novel interactor of the Mph1 helicase in Saccharomyces cerevisiae. In vitro, Mte1 (Mph1-associated telomere maintenance protein 1) binds directly to DNA with a preference for branched molecules such as D loops and fork structures. In addition, Mte1 stimulates the helicase and fork regression activities of Mph1 while inhibiting the ability of Mph1 to dissociate recombination intermediates. Deletion of MTE1 reduces crossover recombination and suppresses the sensitivity of mph1Δ mutant cells to replication stress. Mph1 and Mte1 interdependently colocalize at DNA damage-induced foci and dysfunctional telomeres, and MTE1 deletion results in elongated telomeres. Taken together, our data indicate that Mte1 plays a role in regulation of crossover recombination, response to replication stress, and telomere maintenance.

Getting in (and out of) the loop: regulating higher order telomere structures.

The DNA at the ends of linear chromosomes (the telomere) folds back onto itself and forms an intramolecular lariat-like structure. Although the telomere loop has been implicated in the protection of chromosome ends from nuclease-mediated resection and unscheduled DNA repair activities, it potentially poses an obstacle to the DNA replication machinery during S-phase. Therefore, the coordinated regulation of telomere loop formation, maintenance, and resolution is required in order to establish a balance between protecting the chromosome ends and promoting their duplication prior to cell division. Until recently, the only factor known to influence telomere looping in human cells was TRF2, a component of the shelterin complex. Recent work in yeast and mouse cells has uncovered additional regulatory factors that affect the loop structure at telomeres. In the following "perspective" we outline what is known about telomere looping and highlight the latest results regarding the regulation of this chromosome end structure. We speculate about how the manipulation of the telomere loop may have therapeutic implications in terms of diseases associated with telomere dysfunction and uncontrolled proliferation.

FANCM regulates DNA chain elongation and is stabilized by S-phase checkpoint signalling.

FANCM binds and remodels replication fork structures in vitro. We report that in vivo, FANCM controls DNA chain elongation in an ATPase-dependent manner. In the presence of replication inhibitors that do not damage DNA, FANCM counteracts fork movement, possibly by remodelling fork structures. Conversely, through damaged DNA, FANCM promotes replication and recovers stalled forks. Hence, the impact of FANCM on fork progression depends on the underlying hindrance. We further report that signalling through the checkpoint effector kinase Chk1 prevents FANCM from degradation by the proteasome after exposure to DNA damage. FANCM also acts in a feedback loop to stabilize Chk1. We propose that FANCM is a ringmaster in the response to replication stress by physically altering replication fork structures and by providing a tight link to S-phase checkpoint signalling.

The AAA-ATPase FIGL-1 controls mitotic progression, and its levels are regulated by the CUL-3MEL-26 E3 ligase in the C. elegans germ line.

Members of the AAA-ATPase (ATPases associated with diverse cellular activities) family use the energy from ATP hydrolysis to disrupt protein complexes involved in many cellular processes. Here, we report that FIGL-1 (Fidgetin-like 1), the single Caenorhabditis elegans homolog of mammalian fidgetin and fidgetin-like 1 AAA-ATPases, controls progression through mitosis in the germ line and the early embryo. Loss of figl-1 function leads to the accumulation of mitotic nuclei in the proliferative zone of the germ line, resulting in sterility owing to depletion of germ cells. Like the AAA-ATPase MEI-1 (also known as katanin), FIGL-1 interacts with microtubules and with MEL-26, a specificity factor of CUL-3-based E3 ligases involved in targeting proteins for ubiquitin-dependent degradation by the 26S proteasome. In the germ line, FIGL-1 is enriched in nuclei of mitotic cells, but it disappears at the transition into meiosis. Conversely, MEL-26 expression is low in nuclei of the mitotic zone and induced during meiosis. FIGL-1 accumulates in the germ line and spreads to the meiotic zone after inactivation of mel-26 or cul-3 in vivo. We conclude that degradation of FIGL-1 by the CUL-3MEL-26 E3 ligase spatially restricts FIGL-1 function to mitotic cells, where it is required for correct progression through mitosis.