Biology subjects

Alcon, P.

Publications and source records attributed to Alcon, P..

3 recordsLinked to original sources

bioRxiv2023

The histone chaperone activity of SPT2 controls chromatin structure and function in Metazoa

Histone chaperones control nucleosome density and chromatin structure. In yeast, the H3-H4 chaperone Spt2 controls histone deposition at active genes but its roles in metazoan chromatin structure and organismal physiology are not known. Here we identify the Caenorhabditis elegans orthologue of SPT2 (CeSPT-2) and show that its ability to bind histones H3-H4 is important for germline development and transgenerational epigenetic gene silencing, and that spt-2 mutants display signatures of a global stress response. Genome-wide profiling showed that CeSPT-2 binds to a range of highly expressed genes, and we find that spt-2 mutants have increased chromatin accessibility at these loci. We also show that human SPT2 controls the incorporation of new H3.3 into chromatin. Our work reveals roles for SPT2 in controlling chromatin structure and function in Metazoa.

molecular biology↗

bioRxiv2019

FANCD2-FANCI is a clamp stabilized on DNA by monoubiquitination during DNA repair

Vertebrate DNA crosslink repair is a crucial process that excises toxic replication-blocking DNA interstrand crosslinks1. This pathway fails in the inherited human disease Fanconi anemia (FA), resulting in abnormal development, loss of blood production, and marked cancer susceptibility2. Numerous factors involved in crosslink repair have been identified, and mutations in their corresponding genes cause FA3. A biochemical description of how FA gene products might initiate the removal of DNA crosslinks is now emerging. However, structural insight into this vital process has been limited. Here, we use electron cryomicroscopy (cryoEM) to determine the structure of a key DNA crosslink repair factor - FANCD2 heterodimerized with FANCI (D2-I). Recombinant chicken D2-I adopts a conformation that is in agreement with the structure of its murine counter-part4. In contrast, the activated monoubiquitinated form of D2-I (ubD2-I) adopts an alternative closed conformation, creating a channel that encloses double-stranded DNA. Ubiquitin is positioned at the interface of FANCD2 and FANCI, and acts as a molecular pin to trap ubD2-I in the closed conformation, clamped on DNA. The new solvent-exposed interface around the monoubiquitination site is likely a platform for recruitment of other DNA repair factors. We also find that isolated FANCD2 is a dimer, but it adopts a closed conformation that is unable to bind DNA. When incubated with free FANCI, the FANCD2 homodimer exchanges to D2-I, acquiring DNA binding properties. This suggests an autoinhibitory mechanism that prevents FANCD2 activation on DNA until after assembly with FANCI. Together, our cryoEM and biochemical analysis suggests that D2-I is a clamp that is locked onto DNA by ubiquitin, and provides unanticipated new insight into the regulation and the initiation of DNA cross-link repair.

molecular biology↗

bioRxiv2017

Structure Of The Cpf1 Endonuclease R-Loop Complex After Target DNA Cleavage

Cpf1 is a single RNA-guided endonuclease of class 2 type V CRISPR-Cas system, emerging as a powerful genome editing tool 1,2. To provide insight into its DNA targeting mechanism, we have determined the crystal structure of Francisella novicida Cpf1 (FnCpf1) in complex with the triple strand R-loop formed after target DNA cleavage. The structure reveals a unique machinery for target DNA unwinding to form a crRNA-DNA hybrid and a displaced DNA strand inside FnCpf1. The protospacer adjacent motif (PAM) is recognised by the PAM interacting (PI) domain. In this domain, the conserved K667, K671 and K677 are arranged in a dentate manner in a loop-lysine helix-loop motif (LKL). The helix is inserted at a 45{degrees} angle to the dsDNA longitudinal axis. Unzipping of the dsDNA in a cleft arranged by acidic and hydrophobic residues facilitates the hybridization of the target DNA strand with crRNA. K667 initiates unwinding by pushing away the guanine after the PAM sequence of the dsDNA. The PAM ssDNA is funnelled towards the nuclease site, which is located 70 [A] away, through a hydrophobic protein cavity with basic patches that interact with the phosphate backbone. In this catalytically active conformation the PI and the helix-loop-helix (HLH) motif in the REC1 domain adopt a \"rail shape\" and \"flap-on\" conformations, channelling the PAM strand into the cavity. A steric barrier between the RuvC-II and REC1 domains forms a \"septum\" that separates the displaced PAM strand and the crRNA-DNA hybrid, avoiding re-annealing of the DNA. Mutations in key residues reveal a novel mechanism to determine the DNA product length, thereby linking the PAM and DNAase sites. Our study reveals a singular working model of RNA-guided DNA cleavage by Cpf1, opening up new avenues for engineering this genome modification system2-4.

biochemistry↗