A Role for the Mre11-Rad50-Xrs2 Complex in Gene Expression and Chromosome Organization.

Mre11-Rad50-Xrs2 (MRX) is a highly conserved complex with key roles in various aspects of DNA repair. Here, we report a new function for MRX in limiting transcription in budding yeast. We show that MRX interacts physically and colocalizes on chromatin with the transcriptional co-regulator Mediator. MRX restricts transcription of coding and noncoding DNA by a mechanism that does not require the nuclease activity of Mre11. MRX is required to tether transcriptionally active loci to the nuclear pore complex (NPC), and it also promotes large-scale gene-NPC interactions. Moreover, MRX-mediated chromatin anchoring to the NPC contributes to chromosome folding and helps to control gene expression. Together, these findings indicate that MRX has a role in transcription and chromosome organization that is distinct from its known function in DNA repair.

Mec1 Is Activated at the Onset of Normal S Phase by Low-dNTP Pools Impeding DNA Replication.

The Mec1 and Rad53 kinases play a central role during acute replication stress in budding yeast. They are also essential for viability in normal growth conditions, but the signal that activates the Mec1-Rad53 pathway in the absence of exogenous insults is currently unknown. Here, we show that this pathway is active at the onset of normal S phase because deoxyribonucleotide triphosphate (dNTP) levels present in G1 phase may not be sufficient to support processive DNA synthesis and impede DNA replication. This activation can be suppressed experimentally by increasing dNTP levels in G1 phase. Moreover, we show that unchallenged cells entering S phase in the absence of Rad53 undergo irreversible fork collapse and mitotic catastrophe. Together, these data indicate that cells use suboptimal dNTP pools to detect the onset of DNA replication and activate the Mec1-Rad53 pathway, which in turn maintains functional forks and triggers dNTP synthesis, allowing the completion of DNA replication.

MRX Increases Chromatin Accessibility at Stalled Replication Forks to Promote Nascent DNA Resection and Cohesin Loading.

The recovery of stalled replication forks depends on the controlled resection of nascent DNA and on the loading of cohesin. These processes operate in the context of nascent chromatin, but the impact of nucleosome structure on a fork restart remains poorly understood. Here, we show that the Mre11-Rad50-Xrs2 (MRX) complex acts together with the chromatin modifiers Gcn5 and Set1 and the histone remodelers RSC, Chd1, and Isw1 to promote chromatin remodeling at stalled forks. Increased chromatin accessibility facilitates the resection of nascent DNA by the Exo1 nuclease and the Sgs1 and Chl1 DNA helicases. Importantly, increased ssDNA promotes the recruitment of cohesin to arrested forks in a Scc2-Scc4-dependent manner. Altogether, these results indicate that MRX cooperates with chromatin modifiers to orchestrate the action of remodelers, nucleases, and DNA helicases, promoting the resection of nascent DNA and the loading of cohesin, two key processes involved in the recovery of arrested forks.

RNase H2 degrades toxic RNA:DNA hybrids behind stalled forks to promote replication restart.

R-loops represent a major source of replication stress, but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest, and restart in Saccharomyces cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H-deficient cells could replicate their chromosomes normally under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Treated cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative intermediate in resuming replication. Similar impairments in nascent DNA resection and ssDNA formation at HU-arrested forks were observed in human cells lacking RNase H2. However, fork resection was fully restored by addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.

SAMHD1 acts at stalled replication forks to prevent interferon induction.

SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.

Mrc1 and Rad9 cooperate to regulate initiation and elongation of DNA replication in response to DNA damage.

The S-phase checkpoint maintains the integrity of the genome in response to DNA replication stress. In budding yeast, this pathway is initiated by Mec1 and is amplified through the activation of Rad53 by two checkpoint mediators: Mrc1 promotes Rad53 activation at stalled forks, and Rad9 is a general mediator of the DNA damage response. Here, we have investigated the interplay between Mrc1 and Rad9 in response to DNA damage and found that they control DNA replication through two distinct but complementary mechanisms. Mrc1 rapidly activates Rad53 at stalled forks and represses late-firing origins but is unable to maintain this repression over time. Rad9 takes over Mrc1 to maintain a continuous checkpoint signaling. Importantly, the Rad9-mediated activation of Rad53 slows down fork progression, supporting the view that the S-phase checkpoint controls both the initiation and the elongation of DNA replication in response to DNA damage. Together, these data indicate that Mrc1 and Rad9 play distinct functions that are important to ensure an optimal completion of S phase under replication stress conditions.

Development of a multiple-locus variable-number tandem-repeat typing scheme for genetic fingerprinting of Burkholderia cenocepacia and application to nationwide epidemiological analysis.

Organisms of the Burkholderia cepacia complex are especially important pathogens in cystic fibrosis (CF), with a propensity for patient-to-patient spread and long-term respiratory colonization. B. cenocepacia and Burkholderia multivorans account for the majority of infections in CF, and major epidemic clones have been recognized throughout the world. The aim of the present study was to develop and evaluate a multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) scheme for B. cenocepacia. Potential VNTR loci were identified upon analysis of the annotated genome sequences of B. cenocepacia strains AU1054, J2315, and MCO-3, and 10 of them were selected on the basis of polymorphisms and size. A collection of 100 B. cenocepacia strains, including epidemiologically related and unrelated strains, as well as representatives of the major epidemic lineages, was used to evaluate typeability, epidemiological concordance, and the discriminatory power of MLVA-10 compared with those of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Longitudinal stability was assessed by testing 39 successive isolates from 14 patients. Typeability ranged from 0.91 to 1, except for that of one marker, which was not amplified in 53% of the B. cenocepacia IIIA strains. The MLVA types were shown to be stable in chronically colonized patients and within outbreak-related strains, with excellent epidemiological concordance. Epidemic and/or globally distributed lineages (epidemic Edinburgh-Toronto electrophoretic type 12 [ET-12], sequence type 32 [ST-32], ST-122, ST-234, and ST-241) were successfully identified. Conversely, the discriminatory power of MLVA was lower than that of PFGE or MLST, although PFGE variations within the epidemic lineages sometimes masked their genetic relatedness. In conclusion, MLVA represents a promising cost-effective first-line tool in B. cenocepacia surveillance.