Biology subjects

Gallina, I.

Publications and source records attributed to Gallina, I..

3 recordsLinked to original sources

bioRxiv2023

Catalytic and non-catalytic functions of DNA polymerase kappa in translesion DNA synthesis

Translesion DNA synthesis (TLS) is an essential process that allows cells to bypass lesions encountered during DNA replication and is emerging as a primary target of chemotherapy. Among vertebrate DNA polymerases, polymerase kappa (Pol() has the unique ability to bypass minor groove DNA adducts in vitro. However, Pol(is also required for cells to overcome major groove DNA adducts but the basis of this requirement is unclear. Here, we combine CRISPR base editor screening technology in human cells with TLS analysis of defined DNA lesions in Xenopus egg extracts to unravel the functions and regulations of Pol(during lesion bypass. Strikingly, we show that Pol(has two main functions during TLS, which are differentially regulated via Rev1 binding. On the one hand, Pol(is essential to replicate across minor groove DNA lesions in a process that depends on PCNA ubiquitylation but is independent of Rev1. On the other hand, via its cooperative interaction with Rev1 and ubiquitylated PCNA, Pol(stabilizes the Rev1-Pol(extension complex on DNA to allow extension past major groove DNA lesions and abasic sites, in a process that is independent of Pol(catalytic activity. Together, our work identifies catalytic and non-catalytic functions of Pol(in TLS and reveals important regulatory mechanisms underlying the unique domain architecture present at the C-terminal end of Y-family TLS polymerases.

molecular biology↗

bioRxiv2020

AAV Ablates Neurogenesis in the Adult Murine Hippocampus

Recombinant adeno-associated virus (rAAV) has been widely used as a viral vector across mammalian biology and has been shown to be safe and effective in human gene therapy. We demonstrate that neural progenitor cells (NPCs) and immature dentate granule cells (DGCs) within the adult murine hippocampus are particularly sensitive to rAAV-induced cell death. Cell loss is dose dependent and nearly complete at experimentally relevant viral titers. rAAV-induced cell death is rapid and persistent, with loss of BrdU-labeled cells within 18 hours post-injection and no evidence of recovery of adult neurogenesis at 3 months post-injection. The remaining mature DGCs appear hyperactive 4 weeks post-injection based on immediate early gene expression, consistent with previous studies investigating the effects of attenuating adult neurogenesis. In vitro application of AAV or electroporation of AAV2 inverted terminal repeats (ITRs) is sufficient to induce cell death. Efficient transduction of the dentate gyrus (DG)--without ablating adult neurogenesis--can be achieved by injection of rAAV2-retro serotyped virus into CA3. rAAV2-retro results in efficient retrograde labeling of mature DGCs and permits in vivo 2-photon calcium imaging of dentate activity while leaving adult neurogenesis intact. These findings expand on recent reports implicating rAAV-linked toxicity in stem cells and other cell types and suggest that future work using rAAV as an experimental tool in the DG and as a gene therapy for diseases of the central nervous system (CNS) should be carefully evaluated.

neuroscience↗

bioRxiv2018

Mechanism of replication-coupled DNA-protein crosslink proteolysis by SPRTN and the proteasome

DNA-protein crosslinks (DPCs) are bulky DNA lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S-phase removal of DPCs, but how SPRTN activity is coupled to DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is triggered by single-stranded DNA, a byproduct of DNA replication. In contrast, SPRTN-mediated DPC degradation is independent of DPC polyubiquitylation but requires polymerase extension of a nascent strand to the lesion. Thus, SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms and together promote replication across immovable protein barriers.\n\nHighlightsO_LIThe proteasome, in addition to SPRTN, degrades DPCs during DNA replication\nC_LIO_LIProteasome-dependent DPC degradation requires DPC ubiquitylation\nC_LIO_LIDPC ubiquitylation is triggered by ssDNA and does not require the replisome\nC_LIO_LISPRTN-dependent DPC degradation is a post-replicative process\nC_LI

molecular biology↗