Telomere Length Determines TERRA and R-Loop Regulation through the Cell Cycle.

Maintenance of a minimal telomere length is essential to prevent cellular senescence. When critically short telomeres arise in the absence of telomerase, they can be repaired by homology-directed repair (HDR) to prevent premature senescence onset. It is unclear why specifically the shortest telomeres are targeted for HDR. We demonstrate that the non-coding RNA TERRA accumulates as HDR-promoting RNA-DNA hybrids (R-loops) preferentially at very short telomeres. The increased level of TERRA and R-loops, exclusively at short telomeres, is due to a local defect in RNA degradation by the Rat1 and RNase H2 nucleases, respectively. Consequently, the coordination of TERRA degradation with telomere replication is altered at shortened telomeres. R-loop persistence at short telomeres contributes to activation of the DNA damage response (DDR) and promotes recruitment of the Rad51 recombinase. Thus, the telomere length-dependent regulation of TERRA and TERRA R-loops is a critical determinant of the rate of replicative senescence.

Hold on Tight: Lagging-Strand DNA Polymerases Synthesize Multiple Okazaki Fragments without Letting Go.

Kapadia et al. (2020) use an innovative single-molecule imaging approach in yeast cells to measure chromatin association of individual replisome subunits, thereby challenging the notion that lagging-strand DNA polymerases frequently dissociate from replisomes during DNA replication in vivo.

Molecular and physiological consequences of faulty eukaryotic ribonucleotide excision repair.

The duplication of the eukaryotic genome is an intricate process that has to be tightly safe-guarded. One of the most frequently occurring errors during DNA synthesis is the mis-insertion of a ribonucleotide instead of a deoxyribonucleotide. Ribonucleotide excision repair (RER) is initiated by RNase H2 and results in error-free removal of such mis-incorporated ribonucleotides. If left unrepaired, DNA-embedded ribonucleotides result in a variety of alterations within chromosomal DNA, which ultimately lead to genome instability. Here, we review how genomic ribonucleotides lead to chromosomal aberrations and discuss how the tight regulation of RER timing may be important for preventing unwanted DNA damage. We describe the structural impact of unrepaired ribonucleotides on DNA and chromatin and comment on the potential consequences for cellular fitness. In the context of the molecular mechanisms associated with faulty RER, we have placed an emphasis on how and why increased levels of genomic ribonucleotides are associated with severe autoimmune syndromes, neuropathology, and cancer. In addition, we discuss therapeutic directions that could be followed for pathologies associated with defective removal of ribonucleotides from double-stranded DNA.

The Replisome-Coupled E3 Ubiquitin Ligase Rtt101Mms22 Counteracts Mrc1 Function to Tolerate Genotoxic Stress.

Faithful DNA replication and repair requires the activity of cullin 4-based E3 ubiquitin ligases (CRL4), but the underlying mechanisms remain poorly understood. The budding yeast Cul4 homologue, Rtt101, in complex with the linker Mms1 and the putative substrate adaptor Mms22 promotes progression of replication forks through damaged DNA. Here we characterized the interactome of Mms22 and found that the Rtt101(Mms22) ligase associates with the replisome progression complex during S-phase via the amino-terminal WD40 domain of Ctf4. Moreover, genetic screening for suppressors of the genotoxic sensitivity of rtt101Δ cells identified a cluster of replication proteins, among them a component of the fork protection complex, Mrc1. In contrast to rtt101Δ and mms22Δ cells, mrc1Δ rtt101Δ and mrc1Δ mms22Δ double mutants complete DNA replication upon replication stress by facilitating the repair/restart of stalled replication forks using a Rad52-dependent mechanism. Our results suggest that the Rtt101(Mms22) E3 ligase does not induce Mrc1 degradation, but specifically counteracts Mrc1's replicative function, possibly by modulating its interaction with the CMG (Cdc45-MCM-GINS) complex at stalled forks.

Genetic requirements for repair of lesions caused by single genomic ribonucleotides in S phase.

Single ribonucleoside monophosphates (rNMPs) are transiently present in eukaryotic genomes. The RNase H2-dependent ribonucleotide excision repair (RER) pathway ensures error-free rNMP removal. In some pathological conditions, rNMP removal is impaired. If these rNMPs hydrolyze during, or prior to, S phase, toxic single-ended double-strand breaks (seDSBs) can occur upon an encounter with replication forks. How such rNMP-derived seDSB lesions are repaired is unclear. We expressed a cell cycle phase restricted allele of RNase H2 to nick at rNMPs in S phase and study their repair. Although Top1 is dispensable, the RAD52 epistasis group and Rtt101Mms1-Mms22 dependent ubiquitylation of histone H3 become essential for rNMP-derived lesion tolerance. Consistently, loss of Rtt101Mms1-Mms22 combined with RNase H2 dysfunction leads to compromised cellular fitness. We refer to this repair pathway as nick lesion repair (NLR). The NLR genetic network may have important implications in the context of human pathologies.

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.