Biology subjects

Galanti, L.

Publications and source records attributed to Galanti, L..

4 recordsLinked to original sources

bioRxiv2026

TRF2 mediates force-resistant bridging of distal telomeric DNA repeats

Shelterin binds telomeres, regulates telomere length, and protects chromosome ends from mis-repair, which is essential for genome stability. Telomere end-protection uniquely depends on the TRF2 subunit, which assembles telomeric DNA into a protective T-loop structure in vivo, yet the biochemical mechanism underlying this process remains unresolved. Using single-molecule optical tweezers and atomic force microscopy, we show that the human shelterin complex tethers distal telomeric DNA repeats, looping the intervening DNA. These bridges withstand tens of piconewtons of applied load, rupture at a force that scales with telomeric repeat number and re-form on relaxation. Structure-function analysis demonstrates that this force-resistant tethering is TRF2-dependent under our conditions and depends on multiple interfaces, with the TRFH domain making a major but non-exclusive contribution: introducing the TRF2 TRFH domain into TRF1 is sufficient to confer tethering, whereas its deletion from TRF2 reduces but does not abolish activity. We propose telomeric dsDNA tethering by TRF2 as a candidate first step in T-loop formation, preceding invasion of the telomeric 3' ssDNA overhang.

biophysics↗

bioRxiv2024

Dbf4-Dependent Kinase Finetunes INO80 Function at Chromosome Replication Origins

The highly conserved Dbf4-Dependent Kinase (DDK) plays a pivotal role in the nucleus during S phase, where it directly phosphorylates the replicative helicase, the minichromosome maintenance (MCM) complex. This leads to the initiation of chromosome replication. However, aside from the MCM complex, few other targets have been identified to date, leaving DDK an understudied kinase. Here, we describe a two-pronged mass spectrometry-based approach and define the nuclear DDK-dependent phosphoproteome, which consists of approximately 400 phosphorylation events. Within this network, we found that DDK directly phosphorylates the Arp8 subunit of the multi-subunit chromatin remodeler complex INO80. Arp8 phosphorylation stabilises INO80s intramolecular complex integrity, which finetunes its nucleosome spacing activity at replication origins. This adjustment of origin chromatin architecture stimulates replication and is important for the response to replication stress. Our results represent a significant advance in our understanding of the molecular mechanisms underlying the regulation of replication origins.

biochemistry↗

bioRxiv2021

Pheniqs 2.0: accurate, high performance Bayesian decoding and confidence estimation for combinatorial barcode indexing

BackgroundSystems biology increasingly relies on deep sequencing with combinatorial index tags to associate biological sequences with their sample, cell, or molecule of origin. Accurate data interpretation depends on the ability to classify sequences based on correct decoding of these combinatorial barcodes. The probability of correct decoding is influenced by both sequence quality and the number and arrangement of barcodes. The rising complexity of experimental designs calls for a probability model that accounts for both sequencing errors and random noise, generalizes to multiple combinatorial tags, and can handle any barcoding scheme. The needs for reproducibility and community benchmark standards demand a peer-reviewed tool that preserves decoding quality scores and provides tunable control over classification confidence that balances precision and recall. Moreover, continuous improvements in sequencing throughput require a fast, parallelized and scalable implementation. ResultsWe developed a flexible, robustly engineered software that performs probabilistic decoding and supports arbitrarily complex barcoding designs. Pheniqs computes the full posterior decoding error probability of observed barcodes by consulting basecalling quality scores and prior distributions, and reports sequences and confidence scores in Sequence Alignment/Map (SAM) fields. The product of posteriors for multiple independent barcodes provides an overall confidence score for each read. Pheniqs achieves greater accuracy than minimum edit distance or simple maximum likelihood estimation, and it scales linearly with core count to enable the classification of >11 billion reads in 1h15m using <50 megabytes of memory. Pheniqs has been in production use for seven years in our genomics core facility. ConclusionsWe introduce a computationally efficient software that implements both probabilistic and minimum distance decoders and show that decoding barcodes using posterior probabilities is more accurate than available methods. Pheniqs allows fine-tuning of decoding sensitivity using intuitive confidence thresholds and is extensible with alternative decoders and new error models. Any arbitrary arrangement of barcodes is easily configured, enabling computation of combinatorial confidence scores for any barcoding strategy. An optimized multithreaded implementation assures that Pheniqs is faster and scales better with complex barcode sets than existing tools. Support for POSIX streams and multiple sequencing formats enables easy integration with automated analysis pipelines.

bioinformatics↗

bioRxiv2017

Pheniqs: Fast and flexible quality-aware sequence demultiplexing

1MotivationOutput from high throughput sequencing instruments often exceeds what is necessary to assay a single sample. To better utilize this capacity, multiple samples are independently tagged with a unique \"barcode\" sequence and are then pooled, or \"multiplexed\", and sequenced together. Classifying, or \"demultiplexing\", the reads involves decoding the barcode sequence. Although instruments estimate the probability of incorrectly calling each nucleobase, available demultiplexers do not consult those estimates or report classification error probabilities.\n\nResultsWe present Pheniqs, a fast and flexible sequence demultiplexer and quality analyzer. In addition to providing an efficient implementation of the widespread minimum distance decoder, Pheniqs introduces a novel Phred-adjusted maximum likelihood decoder that consults base calling quality scores and estimates the probability of a barcode decoding error. Setting an upper bound on the permissible error provides an intuitive way to control demultiplexing confidence and directly influence precision and recall. Pheniqs supports FASTQ and multiple Sequence Alignment/Map formats and uses auxiliary SAM tags to report both library classification and demultiplexing error probability. Evaluation on both real and semi-synthetic data indicates that Pheniqs is faster than existing demultiplexers, substantially when demultiplexing longer reads, and achieves greater accuracy by correctly reflecting quality measurements.\n\nAvailability and ImplementationImplemented in multithreaded C++ and available under the terms of the AGPL-3.0 license agreement at http://github.com/biosails/pheniqs. Manual and examples are available at http://biosails.github.io/pheniqs.

bioinformatics↗