Nagaraja Chappidi Group

DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.

Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show that ROS generated by exposure of human cells to the ribonucleotide reductase inhibitor hydroxyurea (HU) promote replication fork reversal in a manner dependent on active transcription and formation of co-transcriptional RNA:DNA hybrids (R-loops). The frequency of R-loop-dependent fork stalling events is also increased after TIMELESS depletion or a partial inhibition of replicative DNA polymerases by aphidicolin, suggesting that this phenomenon is due to a global replication slowdown. In contrast, replication arrest caused by HU-induced depletion of deoxynucleotides does not induce fork reversal but, if allowed to persist, leads to extensive R-loop-independent DNA breakage during S-phase. Our work reveals a link between oxidative stress and transcription-replication interference that causes genomic alterations recurrently found in human cancer.

Recognition of pathogen-derived foreign nucleic acids is central to innate immune defense. This requires discrimination between structurally highly similar self and nonself nucleic acids to avoid aberrant inflammatory responses as in the autoinflammatory disorder Aicardi-Goutières syndrome (AGS). How vast amounts of self RNA are shielded from immune recognition to prevent autoinflammation is not fully understood. Here, we show that human SAM-domain- and HD-domain-containing protein 1 (SAMHD1), one of the AGS-causing genes, functions as a single-stranded RNA (ssRNA) 3'exonuclease, the lack of which causes cellular RNA accumulation. Increased ssRNA in cells leads to dissolution of RNA-protein condensates, which sequester immunogenic double-stranded RNA (dsRNA). Release of sequestered dsRNA from condensates triggers activation of antiviral type I interferon via retinoic-acid-inducible gene I-like receptors. Our results establish SAMHD1 as a key regulator of cellular RNA homeostasis and demonstrate that buffering of immunogenic self RNA by condensates regulates innate immune responses.

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

Mechanisms controlling DNA resection at sites of damage and affecting genome stability have been the subject of deep investigation, though their complexity is not yet fully understood. Specifically, the regulatory role of post-translational modifications in the localization, stability and function of DNA repair proteins is an important aspect of such complexity.

Besides its role in homologous recombination, the tumor suppressor BRCA2 protects stalled replication forks from nucleolytic degradation. Defective fork stability contributes to chemotherapeutic sensitivity of BRCA2-defective tumors by yet-elusive mechanisms. Using DNA fiber spreading and direct visualization of replication intermediates, we report that reversed replication forks are entry points for fork degradation in BRCA2-defective cells. Besides MRE11 and PTIP, we show that RAD52 promotes stalled fork degradation and chromosomal breakage in BRCA2-defective cells. Inactivation of these factors restores reversed fork frequency and chromosome integrity in BRCA2-defective cells. Conversely, impairing fork reversal prevents fork degradation, but increases chromosomal breakage, uncoupling fork protection, and chromosome stability. We propose that BRCA2 is dispensable for RAD51-mediated fork reversal, but assembles stable RAD51 nucleofilaments on regressed arms, to protect them from degradation. Our data uncover the physiopathological relevance of fork reversal and illuminate a complex interplay of homologous recombination factors in fork remodeling and stability.BRCA2 is involved in both homologous recombination (HR) and the protection of stalled replication forks from degradation. Here the authors reveal how HR factors cooperate in fork remodeling, showing that BRCA2 supports RAD51 loading on the regressed arms of reversed replication forks to protect them from degradation.

The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.

Ataxia telangiectasia-mutated and Rad3-related (ATR) protein kinase, a master regulator of DNA-damage response, is activated by RPA-coated single-stranded DNA (ssDNA) generated at stalled replication forks or DNA double-strand breaks (DSBs). Here, we identify the mismatch-binding protein MutSβ, a heterodimer of MSH2 and MSH3, as a key player in this process. MSH2 and MSH3 form a complex with ATR and its regulatory partner ATRIP, and their depletion compromises the formation of ATRIP foci and phosphorylation of ATR substrates in cells responding to replication-associated DSBs. Purified MutSβ binds to hairpin loop structures that persist in RPA-ssDNA complexes and promotes ATRIP recruitment. Mutations in the mismatch-binding domain of MSH3 abolish the binding of MutSβ to DNA hairpin loops and its ability to promote ATR activation by ssDNA. These results suggest that hairpin loops might form in ssDNA generated at sites of DNA damage and trigger ATR activation in a process mediated by MutSβ.

We investigated effects of sodium nitroprusside (SNP) on sucrose metabolizing enzymes, acid, and alkaline invertase and alkaline phosphatase in lemongrass (Cymbopogon flexuosus Steud) Wats varieties i.e. Krishna, Cauveri, Nima and Cheerharit. Fifteen day old lemongrass tillers were treated with SNP (1 and 2 mM) under sunlight for four hours. Our results clearly indicated that SNP (2 mM) substantially decreased the amount of proteins in all varieties studied, with maximum values of 40% and 33% in Nima and Krishna, respectively. SNP (1 mM) significantly increased the amount of proteins 43% and 31% in Krishna and Cauveri, respectively. SNP (2 mM) rapidly and severely reduced the activity of acid and alkaline invertases in all varieties, except Krishna and Cauveri. However, the effect of SNP was more pronounced on acid invertase causing at 2 mM an inhibition of 37%, 35% and 28% in Cheerharit, Nima and Cauveri, respectively, whereas it showed relatively less inhibition in alkaline invertase activity 27%, 24% and 21%, respectively, in Nima, Krishna and Cheerharit. Alkaline phosphatase activity was only considerably decreased following SNP (2 mM) treatment in all lemongrass varieties studied with the exception of Nima, where a sharp decrease of 50% was observed. SNP (1 mM) also demonstrated similar effects on acid and alkaline invertases and alkaline phosphatase. These results clearly suggest that SNP affects acid and alkaline phosphatase activity and, therefore, has a role in sucrose metabolism in lemongrass. Alterations in alkaline phosphatase activity upon SNP treatment have several consequences.