Biology subjects

Moreno, A. T.

Publications and source records attributed to Moreno, A. T..

4 recordsLinked to original sources

bioRxiv2026

Sequential division of labor between PAXX and XLF drives NHEJ synaptic complex stability and maturation

DNA double-strand breaks (DSBs) are primarily repaired by non-homologous end joining (NHEJ), which tethers and ligates DNA ends within a synaptic complex. PAXX is an NHEJ accessory factor related to XRCC4 and XLF, but its mechanistic role and genetic interaction with XLF are unclear. Using biochemical assays and single-molecule imaging within Xenopus laevis egg extracts and human cell assays, we show that PAXX bridges DNA ends by using its disordered tails to bind opposing Ku molecules. This PAXX tether substantially extends long-range synaptic complex (LRSC) lifetime and modestly stabilizes the short-range synaptic complex (SRSC). LRSC stabilization increases XLF residence and promotes the LRSC-to-SRSC transition. Using domain-swapped chimeras, we demonstrate that PAXXs tails provide synaptic stabilization, whereas XLFs heads interact with XRCC4 to drive the LRSC-to-SRSC transition: an XLF head-PAXX tail chimera recapitulates the functions of both proteins, revealing complementary, sequential roles for PAXX and XLF in NHEJ.

biophysics↗

bioRxiv2022

H2A.Z deposition by SWR1C involves multiple ATP-dependent steps

The histone variant H2A.Z is a conserved feature of nucleosomes flanking protein-coding genes. Deposition of H2A.Z requires ATP-dependent replacement of nucleosomal H2A by a chromatin remodeler related to the multi-subunit enzyme, yeast SWR1C. How these enzymes use ATP to promote this nucleosome editing reaction remains unclear. Here we use single-molecule and ensemble methodologies to identify three ATP-dependent phases in the H2A.Z deposition reaction. Real-time analysis of single nucleosome remodeling events reveals an initial, priming step that occurs after ATP addition that likely involves transient DNA unwrapping from the nucleosome. Priming is followed by rapid loss of histone H2A, which is subsequently released from the H2A.Z nucleosomal product. Surprisingly, the rates of both priming and the release of the H2A/H2B dimer are sensitive to ATP concentration. This complex reaction pathway provides multiple opportunities to regulate the timely and accurate deposition of H2A.Z at key genomic locations.

biochemistry↗

bioRxiv2020

XLF acts as a flexible connector during non-homologous end joining

Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here we investigate the role of the intrinsically disordered C-terminal tail of XLF, a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku binding motif (KBM) at the extreme C-terminus are required for end joining. While the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.

biochemistry↗

bioRxiv2019

Non-homologous end joining minimizes errors by coordinating DNA processing with ligation

Genome stability requires efficient and faithful repair of DNA double-strand breaks (DSBs). The predominant DSB repair pathway in human cells is non-homologous end-joining (NHEJ), which directly ligates DNA ends1-5. Broken DNA ends at DSBs are chemically diverse, and many are not compatible for direct ligation by the NHEJ-associated DNA Ligase IV (Lig4). To solve this problem, NHEJ end-processing enzymes including polymerases and nucleases modify ends until they are ligatable. How cells regulate end processing to minimize unnecessary genomic alterations6 during repair of pathological DSBs remains unknown. Using a biochemical system that recapitulates key features of cellular NHEJ, we previously demonstrated that DNA ends are initially tethered at a distance, followed by Lig4-mediated formation of a "short-range synaptic complex" in which DNA ends are closely aligned for ligation7. Here, we show that a wide variety of end-processing activities all depend on formation of the short-range complex. Moreover, using real-time single molecule imaging, we find that end processing occurs within the short-range complex. Confining end processing to the Lig4-dependent short-range synaptic complex promotes immediate ligation of compatible ends and ensures that incompatible ends are ligated as soon as they become compatible, thereby minimizing end processing. Our results elucidate how NHEJ exploits end processing to achieve versatility while minimizing errors that cause genome instability.

molecular biology↗