DNA polymerase theta (POLQ) repairs mitotic DNA breaks; this requires RHINO and PLK1, averts genomic instability and may underlie effects of POLQ inhibitors in HDR-deficient cancer cells. We... Show moreDNA polymerase theta (POLQ) repairs mitotic DNA breaks; this requires RHINO and PLK1, averts genomic instability and may underlie effects of POLQ inhibitors in HDR-deficient cancer cells. We discuss recent work on mitotic DNA break processing and repair, the need for multiple DSB repair pathways and implications of therapeutic POLQ targeting in cancer. Show less
DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the replicative DNA polymerase Pol III... Show moreDNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the replicative DNA polymerase Pol III alpha terminates at a previously synthesized RNA primer, DNA Pol I takes over and continues DNA synthesis while displacing the downstream RNA primer. The displaced primer is subsequently excised by an endonuclease, followed by the sealing of the nick by a DNA ligase. Yet how the sequential actions of Pol III alpha, Pol I polymerase, Pol I endonuclease and DNA ligase are coordinated is poorly defined. Here we show that each enzymatic activity prepares the DNA substrate for the next activity, creating an efficient four-point molecular handover. The cryogenic-electron microscopy structure of Pol I bound to a DNA substrate with both an upstream and downstream primer reveals how it displaces the primer in a manner analogous to the monomeric helicases. Moreover, we find that in addition to its flap-directed nuclease activity, the endonuclease domain of Pol I also specifically cuts at the RNA-DNA junction, thus marking the end of the RNA primer and creating a 5 ' end that is a suitable substrate for the ligase activity of LigA once all RNA has been removed. Show less
DNA mismatch repair detects and corrects mismatches introduced during DNA replication. The protein MutS scans for mismatches and coordinates the repair cascade. During this process, MutS undergoes... Show moreDNA mismatch repair detects and corrects mismatches introduced during DNA replication. The protein MutS scans for mismatches and coordinates the repair cascade. During this process, MutS undergoes multiple conformational changes in response to ATP binding, hydrolysis and release, but how ATP induces the various MutS conformations is incompletely understood. Here we present four cryogenic electron microscopy structures of Escherichia coli MutS at sequential stages of the ATP hydrolysis cycle that reveal how ATP binding and hydrolysis induce closing and opening of the MutS dimer, respectively. Biophysical analysis demonstrates how DNA binding modulates the ATPase cycle by prevention of hydrolysis during scanning and mismatch binding, while preventing ADP release in the sliding clamp state. Nucleotide release is achieved when MutS encounters single-stranded DNA that is produced during removal of the daughter strand. The combination of ATP binding and hydrolysis and its modulation by DNA enables MutS to adopt the different conformations needed to coordinate the sequential steps of the mismatch repair cascade.Cryogenic electron microscopy structures of Escherichia coli MutS at sequential stages of the ATP hydrolysis cycle reveal how ATP binding and hydrolysis and its modulation by DNA enable MutS to adopt different conformations during mismatch repair. Show less
DNA mismatch repair detects and removes mismatches from DNA by a conserved mechanism, reducing the error rate of DNA replication by 100- to 1,000-fold. In this process, MutS homologs scan DNA,... Show moreDNA mismatch repair detects and removes mismatches from DNA by a conserved mechanism, reducing the error rate of DNA replication by 100- to 1,000-fold. In this process, MutS homologs scan DNA, recognize mismatches and initiate repair. How the MutS homologs selectively license repair of a mismatch among millions of matched base pairs is not understood. Here we present four cryo-EM structures of Escherichia coli MutS that provide snapshots, from scanning homoduplex DNA to mismatch binding and MutL activation via an intermediate state. During scanning, the homoduplex DNA forms a steric block that prevents MutS from transitioning into the MutL-bound clamp state, which can only be overcome through kinking of the DNA at a mismatch. Structural asymmetry in all four structures indicates a division of labor between the two MutS monomers. Together, these structures reveal how a small conformational change from the homoduplex- to heteroduplex-bound MutS acts as a licensing step that triggers a dramatic conformational change that enables MutL binding and initiation of the repair cascade.Cryo-EM reconstructions and atomic models reveal the mechanism of MutS-MutL DNA mismatch recognition and repair initiation. Show less
Barcena, M.; Barnes, C.O.; Beck, M.; Bjorkman, P.J.; Canard, B.; Gao, G.R.F.; ... ; Zhou, Q. 2021
How can structural biology help us understand and combat SARS-CoV-2? Researchers in the field share their experiences and opinions and point to the challenges that lie ahead.
Structural, biochemical and cellular analyses show that bacterial antigens can mimic gliadin epitopes involved in celiac disease being presented by HLA-DQ2.5 and recognized by T cells derived from... Show moreStructural, biochemical and cellular analyses show that bacterial antigens can mimic gliadin epitopes involved in celiac disease being presented by HLA-DQ2.5 and recognized by T cells derived from patients.The human leukocyte antigen (HLA) locus is strongly associated with T cell-mediated autoimmune disorders. HLA-DQ2.5-mediated celiac disease (CeD) is triggered by the ingestion of gluten, although the relative roles of genetic and environmental risk factors in CeD is unclear. Here we identify microbially derived mimics of gliadin epitopes and a parental bacterial protein that is naturally processed by antigen-presenting cells and activated gliadin reactive HLA-DQ2.5-restricted T cells derived from CeD patients. Crystal structures of T cell receptors in complex with HLA-DQ2.5 bound to two distinct bacterial peptides demonstrate that molecular mimicry underpins cross-reactivity toward the gliadin epitopes. Accordingly, gliadin reactive T cells involved in CeD pathogenesis cross-react with ubiquitous bacterial peptides, thereby suggesting microbial exposure as a potential environmental factor in CeD. Show less
Xue, X.G.; Wu, J.; Ricklin, D.; Forneris, F.; Crescenzio, P. di; Schmidt, C.Q.; ... ; Gros, P. 2017
