Project description:Fused in sarcoma (FUS) is involved in the regulation of RNA and DNA metabolism. FUS participates in the formation of biomolecular condensates driven by phase transition. FUS is prone to self-aggregation and tends to undergo phase transition both with or without nucleic acid polymers. Using dynamic light scattering and fluorescence microscopy, we examined the formation of FUS high-order structures or FUS-rich microphases induced by the presence of RNA, poly(ADP-ribose), ssDNA, or dsDNA and evaluated effects of some nucleic-acid-binding proteins on the phase behavior of FUS-nucleic acid systems. Formation and stability of FUS-rich microphases only partially correlated with FUS's affinity for a nucleic acid polymer. Some proteins-which directly interact with PAR, RNA, ssDNA, and dsDNA and are possible components of FUS-enriched cellular condensates-disrupted the nucleic-acid-induced assembly of FUS-rich microphases. We found that XRCC1, a DNA repair factor, underwent a microphase separation and formed own microdroplets and coassemblies with FUS in the presence of poly(ADP-ribose). These results probably indicated an important role of nucleic-acid-binding proteins in the regulation of FUS-dependent formation of condensates and imply the possibility of the formation of XRCC1-dependent phase-separated condensates in the cell.
Project description:Cellular DNA repair processes are crucial to maintain genome stability and integrity. In DNA base excision repair, a tight heterodimer complex formed by DNA polymerase β (Polβ) and XRCC1 is thought to facilitate repair by recruiting Polβ to DNA damage sites. Here we show that disruption of the complex does not impact DNA damage response or DNA repair. Instead, the heterodimer formation is required to prevent ubiquitylation and degradation of Polβ. In contrast, the stability of the XRCC1 monomer is protected from CHIP-mediated ubiquitylation by interaction with the binding partner HSP90. In response to cellular proliferation and DNA damage, proteasome and HSP90-mediated regulation of Polβ and XRCC1 alters the DNA repair complex architecture. We propose that protein stability, mediated by DNA repair protein complex formation, functions as a regulatory mechanism for DNA repair pathway choice in the context of cell cycle progression and genome surveillance.
Project description:PAXX is a recently identified component of the nonhomologous end joining (NHEJ) DNA repair pathway. The molecular mechanisms of PAXX action remain largely unclear. Here we characterise the interactomes of PAXX and its paralogs, XLF and XRCC4, to show that these factors share the ability to interact with DNA polymerase λ (Pol λ), stimulate its activity and are required for recruitment of Pol λ to laser-induced DNA damage sites. Stimulation of Pol λ activity by XRCC4 paralogs requires a direct interaction between the SP/8 kDa domain of Pol λ and their N-terminal head domains to facilitate recognition of the 5' end of substrate gaps. Furthermore, PAXX and XLF collaborate with Pol λ to promote joining of incompatible DNA ends and are redundant in supporting Pol λ function in vivo. Our findings identify Pol λ as a novel downstream effector of PAXX function and show XRCC4 paralogs act in synergy to regulate polymerase activity in NHEJ.
Project description:Family X polymerases such as DNA polymerase lambda (Pol lambda) are well suited for filling short gaps during DNA repair because they simultaneously bind both the 5' and 3' ends of short gaps. DNA binding and gap filling are well characterized for 1-nucleotide (nt) gaps, but the location of yet-to-be-copied template nucleotides in longer gaps is unknown. Here we present crystal structures revealing that, when bound to a 2-nt gap, Pol lambda scrunches the template strand and binds the additional uncopied template base in an extrahelical position within a binding pocket that comprises three conserved amino acids. Replacing these amino acids with alanine results in less processive gap filling and less efficient NHEJ when 2-nt gaps are involved. Thus, akin to scrunching by RNA polymerase during transcription initiation, scrunching occurs during gap filling DNA synthesis associated with DNA repair.
Project description:Self-organization of cells is central to a variety of biological systems and physical concepts of condensed matter have proven instrumental in deciphering some of their properties. Here we show that microphase separation, long studied in polymeric materials and other inert systems, has a natural counterpart in living cells. When placed below a millimetric film of liquid nutritive medium, a quasi two-dimensional, high-density population of Dictyostelium discoideum cells spontaneously assembles into compact domains. Their typical size of 100 μm is governed by a balance between competing interactions: an adhesion acting as a short-range attraction and promoting aggregation, and an effective long-range repulsion stemming from aerotaxis in near anoxic condition. Experimental data, a simple model and cell-based simulations all support this scenario. Our findings establish a generic mechanism for self-organization of living cells and highlight oxygen regulation as an emergent organizing principle for biological matter.
Project description:Reactive oxygen species (ROS) interact with DNA, frequently generating highly mutagenic 7,8-dihydro-8-oxoguanine (8-oxo-G) lesions. Replicative DNA polymerases (pols) often misincorporate adenine opposite 8-oxo-G. The subsequent repair mechanism allowing the removal of adenine and formation of C:8-oxo-G base pair is essential to prevent C:G to A:T transversion mutations. Here, we show by immunofluorescence experiments, in cells exposed to ROS, the involvement of MutY glycosylase homologue (MUTYH) and DNA pol lambda in the repair of A:8-oxo-G mispairs. We observe specific recruitment of MUTYH, DNA pol lambda, proliferating cell nuclear antigen (PCNA), flap endonuclease 1 (FEN1) and DNA ligases I and III from human cell extracts to A:8-oxo-G DNA, but not to undamaged DNA. Using purified human proteins and a DNA template, we reconstitute the full pathway for the faithful repair of A:8-oxo-G mispairs involving MUTYH, DNA pol lambda, FEN1, and DNA ligase I. These results reveal a cellular response pathway to ROS, important to sustain genomic stability and modulate carcinogenesis.
Project description:In eukaryotes, DNA is packed inside the cell nucleus in the form of chromatin, which consists of DNA, proteins such as histones, and RNA. Euchromatin, which is permissive for transcription, is spatially organized into transcriptionally inactive domains interspersed with pockets of transcriptional activity. While transcription and RNA have been implicated in euchromatin organization, it remains unclear how their interplay forms and maintains transcription pockets. Here we combine theory and experiment to analyze the dynamics of euchromatin organization as pluripotent zebrafish cells exit mitosis and begin transcription. We show that accumulation of RNA induces formation of transcription pockets which displace transcriptionally inactive chromatin. We propose that the accumulating RNA recruits RNA-binding proteins that together tend to separate from transcriptionally inactive euchromatin. Full phase separation is prevented because RNA remains tethered to transcribed euchromatin through RNA polymerases. Instead, smaller scale microphases emerge that do not grow further and form the typical pattern of euchromatin organization.
Project description:DNA polymerase λ (pol λ) functions in DNA repair with its main roles considered to be filling short gaps during repair of double-strand breaks by nonhomologous end joining and during base excision repair. As indicated by structural and biochemical studies over the past 10 years, pol λ shares many common properties with other family X siblings (pol β, pol μ, and terminal deoxynucleotidyl transferase) but also has unique structural features that determine its specific functions. In this review, we consider how structural studies over the past decade furthered our understanding of the behavior and biological roles of pol λ.
Project description:Scaffold proteins play a central role in DNA repair by recruiting and organizing sets of enzymes required to perform multi-step repair processes. X-ray cross complementing group 1 protein (XRCC1) forms enzyme complexes optimized for single-strand break repair, but participates in other repair pathways as well. Available structural data for XRCC1 interactions is summarized and evaluated in terms of its proposed roles in DNA repair. Mutational approaches related to the abrogation of specific XRCC1 interactions are also discussed. Although substantial progress has been made in elucidating the structural basis for XRCC1 function, the molecular mechanisms of XRCC1 recruitment related to several proposed roles of the XRCC1 DNA repair complex remain undetermined.
Project description:Base excision repair (BER) generates gapped DNA intermediates containing a 5'-terminal 2-deoxyribose-5-phosphate (5'-dRP) group. In mammalian cells, gap filling and dRP removal are catalyzed by Pol β, which belongs to the X family of DNA polymerases. In higher plants, the only member of the X family of DNA polymerases is Pol λ. Although it is generally believed that plant Pol λ participates in BER, there is limited experimental evidence for this hypothesis. Here we have characterized the biochemical properties of Arabidopsis thaliana Pol λ (AtPol λ) in a BER context, using a variety of DNA repair intermediates. We have found that AtPol λ performs gap filling inserting the correct nucleotide, and that the rate of nucleotide incorporation is higher in substrates containing a C in the template strand. Gap filling catalyzed by AtPol λ is most efficient with a phosphate at the 5'-end of the gap and is not inhibited by the presence of a 5'-dRP mimic. We also show that AtPol λ possesses an intrinsic dRP lyase activity that is reduced by mutations at two lysine residues in its 8-kDa domain, one of which is present in Pol λ exclusively and not in any Pol β homolog. Importantly, we also found that the dRP lyase activity of AtPol λ allows efficient completion of uracil repair in a reconstituted short-patch BER reaction. These results suggest that AtPol λ plays an important role in plant BER.