DNA double-strand break (DSB) repair is initiated by DNA end resection. CtIP acts in short-range resection to stimulate MRE11-RAD50-NBS1 (MRN) to endonucleolytically cleave 5'-terminated DNA to bypass protein blocks. CtIP also promotes the DNA2 helicase-nuclease to accelerate long-range resection downstream from MRN. Here, using AlphaFold2, we identified CtIP-F728E-Y736E as a separation-of-function mutant that is still proficient in conjunction with MRN but is not able to stimulate ssDNA degradation by DNA2. Accordingly, CtIP-F728E-Y736E impairs physical interaction with DNA2. Cellular assays revealed that CtIP-F728E-Y736E cells exhibit reduced DSB-dependent chromatin-bound RPA, impaired long-range resection, and increased sensitivity to DSB-inducing drugs. Previously, CtIP was shown to be targeted by PLK1 to inhibit long-range resection, yet the underlying mechanism was unclear. We show that the DNA2-interacting region in CtIP includes the PLK1 target site at S723. The integrity of S723 in CtIP is necessary for the stimulation of DNA2, and phosphorylation of CtIP by PLK1 in vitro is consequently inhibitory, explaining why PLK1 restricts long-range resection. Our data support a model in which CDK-dependent phosphorylation of CtIP activates resection by MRN in S phase, and PLK1-mediated phosphorylation of CtIP disrupts CtIP stimulation of DNA2 to attenuate long-range resection later at G2/M.

The MRN complex (MRX in Saccharomyces cerevisiae, made of Mre11, Rad50 and Nbs1/Xrs2) initiates double-stranded DNA break repair and activates the Tel1/ATM kinase in the DNA damage response. Telomeres counter both outcomes at chromosome ends, partly by keeping MRN-ATM in check. We show that MRX is disabled by telomeric protein Rif2 through an N-terminal motif (MIN, MRN/X-inhibitory motif). MIN executes suppression of Tel1, DNA end-resection and non-homologous end joining by binding the Rad50 N-terminal region. Our data suggest that MIN promotes a transition within MRX that is not conductive for endonuclease activity, DNA-end tethering or Tel1 kinase activation, highlighting an Achilles' heel in MRN, which we propose is also exploited by the RIF2 paralog ORC4 (Origin Recognition Complex 4) in Kluyveromyces lactis and the Schizosaccharomyces pombe telomeric factor Taz1, which is evolutionarily unrelated to Orc4/Rif2. This raises the possibility that analogous mechanisms might be deployed in other eukaryotes as well.

The Mre11-Rad50-Xrs2 (MRX) complex detects and processes DNA double-strand breaks (DSBs). Its DNA binding and processing activities are regulated by transitions between an ATP-bound state and a post-hydrolysis cutting state that is nucleolytically active. Mre11 endonuclease activity is stimulated by Sae2, whose lack increases MRX persistence at DSBs and checkpoint activation. Here we show that the Rif2 protein inhibits Mre11 endonuclease activity and is responsible for the increased MRX retention at DSBs in sae2Δ cells. We identify a Rad50 residue that is important for Rad50-Rif2 interaction and Rif2 inhibition of Mre11 nuclease. This residue is located near a Rad50 surface that binds Sae2 and is important in stabilizing the Mre11-Rad50 (MR) interaction in the cutting state. We propose that Sae2 stimulates Mre11 endonuclease activity by stabilizing a post-hydrolysis MR conformation that is competent for DNA cleavage, whereas Rif2 antagonizes this Sae2 function and stabilizes an endonuclease inactive MR conformation.

Highly active antiretroviral therapy (HAART) significantly reduces HIV-1 replication and prevents progression to AIDS. However, residual low-level viremia (LLV) persists and long-lived viral reservoirs are maintained in anatomical sites. These reservoirs permit a recrudescence of viremia upon cessation of therapy and thus HAART must be maintained indefinitely. HIV-1 reservoirs include latently infected resting memory CD4⁺ T-cells and macrophages which may contribute to residual viremia. It has not been conclusively determined if a component of LLV may also be due to residual replication in cells with sub-therapeutic drug levels and/or long-lived chronically infected cells. In this study, RT-SHIV(mac239) diversity was characterized in five rhesus macaques that received a five-drug HAART regimen [tenofovir, emtricitabine, zidovudine, amdoxovir, (A, C, T, G nucleoside analogs) and the non-nucleoside reverse transcriptase (RT) inhibitor efavirenz]. Before maximal viral load suppression, longitudinal plasma viral RNA RT diversity was analyzed using a 454 sequencer. After suppression, LLV RT diversity (amino acids 65-210) was also assessed. LLV samples had viral levels less than our standard detection limit (50 viral RNA copies/mL) and few transient blips <200 RNA copies/mL. HAART was discontinued in three macaques after 42 weeks of therapy resulting in viral rebound. The level of viral divergence and the prevalence of specific alleles in LLV was similar to pre-suppression viremia. While some LLV sequences contained mutations not observed in the pre-suppression profile, LLV was not characterized by temporal viral evolution or apparent selection of drug resistance mutations. Similarly, resistance mutations were not detected in the viral rebound population. Interestingly, one macaque maintained a putative LLV predominant plasma clone sequence. Together, these results suggest that residual replication did not markedly contribute to LLV and that this model mimics the prevalence and phylogenetic characteristics of LLV during human HAART. Therefore, this model may be ideal for testing HIV-1 eradication strategies.

The Mre11-Rad50-Xrs2/Nbs1 (MRX/N) complex orchestrates the cellular response to DSBs through its structural, enzymatic, and signaling roles. Xrs2/Nbs1 is essential for nuclear translocation of Mre11, but its role as a component of the complex is not well defined. Here, we demonstrate that nuclear localization of Mre11 (Mre11-NLS) is able to bypass several functions of Xrs2, including DNA end resection, meiosis, hairpin resolution, and cellular resistance to clastogens. Using purified components, we show that the MR complex has equivalent activity to MRX in cleavage of protein-blocked DNA ends. Although Xrs2 physically interacts with Sae2, we found that end resection in its absence remains Sae2 dependent in vivo and in vitro. MRE11-NLS was unable to rescue the xrs2Δ defects in Tel1/ATM kinase signaling and non-homologous end joining, consistent with the role of Xrs2 as a chaperone and adaptor protein coordinating interactions between the MR complex and other repair proteins.

DNA end resection plays a critical function in DNA double-strand break repair pathway choice. Resected DNA ends are refractory to end-joining mechanisms and are instead channeled to homology-directed repair. Using biochemical, genetic, and imaging methods, we show that phosphorylation of Saccharomyces cerevisiae Sae2 controls its capacity to promote the Mre11-Rad50-Xrs2 (MRX) nuclease to initiate resection of blocked DNA ends by at least two distinct mechanisms. First, DNA damage and cell cycle-dependent phosphorylation leads to Sae2 tetramerization. Second, and independently, phosphorylation of the conserved C-terminal domain of Sae2 is a prerequisite for its physical interaction with Rad50, which is also crucial to promote the MRX endonuclease. The lack of this interaction explains the phenotype of rad50S mutants defective in the processing of Spo11-bound DNA ends during meiotic recombination. Our results define how phosphorylation controls the initiation of DNA end resection and therefore the choice between the key DNA double-strand break repair mechanisms.

RT-SHIV is a chimera of simian immunodeficiency virus (SIV) containing the reverse transcriptase (RT)-encoding region of human immunodeficiency virus type 1 (HIV-1) within the backbone of SIVmac239. It has been used in a non-human primate model for studies of non-nucleoside RT inhibitors (NNRTI) and highly active antiretroviral therapy (HAART). We and others have identified several mutations that arise in the "foreign" HIV-1 RT of RT-SHIV during in vivo replication. In this study we catalogued amino acid substitutions in the HIV-1 RT and in regions of the SIV backbone with which RT interacts that emerged 30 weeks post-infection from seven RT-SHIV-infected rhesus macaques. The virus set points varied from relatively high virus load, moderate virus load, to undetectable virus load. The G196R substitution in RT was detected from 6 of 7 animals at week 4 post-infection and remained in virus from 4 of 6 animals at week 30. Virus from four high virus load animals showed several common mutations within RT, including L74V or V75L, G196R, L214F, and K275R. The foreign RT from high virus load isolates exhibited as much variation as that of the highly variable envelope surface glycoprotein, and 10-fold higher than that of the native RT of SIVmac239. Isolates from moderate virus load animals showed much less variation in the foreign RT than the high virus load isolates. No variation was found in SIVmac239 genes known to interact with RT. Our results demonstrate substantial adaptation of the foreign HIV-1 RT in RT-SHIV-infected macaques, which most likely reflects selective pressure upon the foreign RT to attain optimal activity within the context of the chimeric RT-SHIV and the rhesus macaque host.

To prevent progression to AIDS, persons infected with human immunodeficiency virus type 1 (HIV-1) must remain on highly active antiretroviral therapy (HAART) indefinitely since this modality does not eradicate the virus. The mechanisms involved in viral persistence during HAART are poorly understood, but an animal model of HAART could help elucidate these mechanisms and enable studies of HIV-1 eradication strategies. Due to the specificity of non-nucleoside reverse transcriptase (RT) inhibitors (NNRTIs) for HIV-1, we have used RT-SHIV, a chimeric virus of simian immunodeficiency virus with RT from HIV-1. This virus is susceptible to NNRTIs and causes an AIDS-like disease in rhesus macaques. In this study, two groups of HAART-treated, RT-SHIV-infected macaques were analyzed to determine viral decay kinetics. In the first group, viral loads were monitored with a standard TaqMan RT-PCR assay with a limit of detection of 50 viral RNA copies per mL. Upon initiation of HAART, viremia decayed in a bi-phasic manner with half-lives of 1.7 and 8.5 days, respectively. A third phase was observed with little further decay. In the second group, the macaques were followed longitudinally with a more sensitive assay utilizing ultracentrifugation to concentrate virus from plasma. Bi-phasic decay of viral RNA was also observed in these animals with half-lives of 1.8 and 5.8 days. Viral loads in these animals during a third phase ranged from 2-58 RNA copies/mL, with little decay over time. The viral decay kinetics observed in these macaques are similar to those reported for HIV-1 infected humans. These results demonstrate that low-level viremia persists in RT-SHIV-infected macaques despite a HAART regimen commonly used in humans.

To repair a DNA double-strand break (DSB) by homologous recombination (HR), the 5'-terminated strand of the DSB must be resected. The human MRE11-RAD50-NBS1 (MRN) and CtIP proteins were implicated in the initiation of DNA end resection, but the underlying mechanism remained undefined. Here, we show that CtIP is a co-factor of the MRE11 endonuclease activity within the MRN complex. This function is absolutely dependent on CtIP phosphorylation that includes the key cyclin-dependent kinase target motif at Thr-847. Unlike in yeast, where the Xrs2/NBS1 subunit is dispensable in vitro, NBS1 is absolutely required in the human system. The MRE11 endonuclease in conjunction with RAD50, NBS1, and phosphorylated CtIP preferentially cleaves 5'-terminated DNA strands near DSBs. Our results define the initial step of HR that is particularly relevant for the processing of DSBs bearing protein blocks.

The Werner Syndrome helicase, WRN, is a promising therapeutic target in cancers with microsatellite instability (MSI). Long-term MSI leads to the expansion of TA nucleotide repeats proposed to form cruciform DNA structures, which in turn cause DNA breaks and cell lethality upon WRN downregulation. Here we employed biochemical assays to show that WRN helicase can efficiently and directly unfold cruciform structures, thereby preventing their cleavage by the SLX1-SLX4 structure-specific endonuclease. TA repeats are particularly prone to form cruciform structures, explaining why these DNA sequences are preferentially broken in MSI cells upon WRN downregulation. We further demonstrate that the activity of the DNA mismatch repair (MMR) complexes MutSα (MSH2-MSH6), MutSβ (MSH2-MSH3), and MutLα (MLH1-PMS2) similarly decreases the level of DNA cruciforms, although the mechanism is different from that employed by WRN. When combined, WRN and MutLα exhibited higher than additive effects in in vitro cruciform processing, suggesting that WRN and the MMR proteins may cooperate. Our data explain how WRN and MMR defects cause genome instability in MSI cells with expanded TA repeats, and provide a mechanistic basis for their recently discovered synthetic-lethal interaction with promising applications in precision cancer therapy.