Biology subjects

Lopezcolorado, F. W.

Publications and source records attributed to Lopezcolorado, F. W..

7 recordsLinked to original sources

bioRxiv2025

53BP1/RIF1 and DNA-PKcs show distinct genetic interactions with diverse chromosomal break repair outcomes.

DNA double strand breaks (DSBs) are the effective lesion of cancer radiotherapy and induce gene editing. 53BP1 accumulates at DSBs and is implicated in end joining (EJ) repair, but its influence on DSB repair is distinct from canonical non-homologous end joining (C-NHEJ). We sought to define the genetic interplay of 53BP1 with C-NHEJ, focusing on the DNA-PKcs kinase. We examined Cas9 DSBs, which largely generates blunt DSBs, since blunt DSB EJ is dependent on C-NHEJ. Loss of 53BP1 does not affect blunt DSB EJ, but causes a reduction in such repair in DNA-PKcs deficient cells. In contrast, disrupting 53BP1 and DNA-PKcs, both alone and together, has similar effects on the type of deletion mutation (increase in microhomology deletions). We found similar effects on EJ with RIF1 loss, which is a downstream effector of 53BP1. Thus, 53BP1/RIF1 appear to play a backup role for DNA-PKcs during blunt DSB EJ, but function in the same pathway to suppress microhomology deletions. In contrast, 53BP1 and DNA-PKcs function independently to suppress homology-directed repair. Finally, DNA-PKcs kinase inhibition causes marked radiosensitivity, which is not additive with loss of 53BP1 and RIF1. Altogether, 53BP1/RIF1 and DNA-PKcs show distinct genetic interactions with diverse DSB repair outcomes.

molecular biology↗

bioRxiv2024

Distinct functions of PAXX and MRI during chromosomal end joining

A key step of Canonical Nonhomologous End Joining (C-NHEJ) is synapsis of DNA double strand break (DSB) ends for ligation. The DNA-PKcs dimer mediates synapsis in a long-range complex with DSB ends remaining apart, whereas the XLF homodimer can mediate synapsis in both long-range and short-range complexes. Recent structural studies found the PAXX homodimer may also facilitate synapsis in long-range complexes with DNA-PKcs via its interactions with Ku70. Thus, we examined the influence of PAXX in C-NHEJ of chromosomal DSBs, which we compared to another Ku-binding factor, MRI. Using EJ of blunt DSBs with Cas9 reporters as a readout for C-NHEJ, we found that PAXX and/or MRI are dispensable. However, when combined with disruption of DNA-PKcs, particularly with DNA-PKcs kinase inhibition, PAXX becomes important for blunt DSB EJ. In contrast, while DNA-PKcs is also important to suppress short deletion mutations with microhomology, this effect is not magnified with PAXX loss. MRI loss had no effect combined with DNA-PKcs disruption, but becomes important for blunt DSB EJ when combined with disruption of XLF, as is PAXX. Finally, XLF loss causes an increase in larger deletions compared to DNA-PKcs inhibition, which is magnified with combined loss of MRI. Altogether, we suggest that PAXX promotes DSB end synapsis during C-NHEJ in a manner that is partially redundant with DNA-PKcs and XLF, whereas MRI appears to be mainly important in the context of XLF disruption. SIGNIFICANCE STATEMENTCanonical Nonhomologous End Joining (C-NHEJ) is a DNA double strand break repair pathway that can repair diverse types of DNA ends, but is prone to causing genetic mutations. In particular, C-NHEJ can cause insertion or deletion mutations that can cause human disease. Therefore, it is critical to understand the mechanisms of C-NHEJ, including and how various factors within the pathway affect repair outcomes. In this study, we elucidate the role of factors PAXX and MRI during chromosomal EJ and their interplay with the C-NHEJ factors DNA-PKcs and XLF.

genetics↗

bioRxiv2024

Functions of PMS2 and MLH1 important for regulation of divergent repeat-mediated deletions

Repeat-mediated deletions (RMDs) are a type of deletion rearrangement that utilizes two repetitive elements to bridge a DNA double-strand break (DSB) that leads to loss of the intervening sequence and one of the repeats. Sequence divergence between repeats causes RMD suppression and indeed this divergence must be resolved in the RMD products. The mismatch repair factor, MLH1, was shown to be critical for both RMD suppression and a polarity of sequence divergence resolution in RMDs. Here, we sought to study the interrelationship between these two aspects of RMD regulation (i.e., RMD suppression and polar divergence resolution), by examining several mutants of MLH1 and its binding partner PMS2. To begin with, we show that PMS2 is also critical for both RMD suppression and polar resolution of sequence divergence in RMD products. Then, with six mutants of the MLH1-PMS2 heterodimer, we found several different patterns: three mutants showed defects in both functions, one mutant showed loss of RMD suppression but not polar divergence resolution, whereas another mutant showed the opposite, and finally one mutant showed loss of RMD suppression but had a complex effect on polar divergence resolution. These findings indicate that RMD suppression vs. polar resolution of sequence divergence are distinct functions of MLH1-PMS2. HIGHLIGHTSO_LIMLH1-PMS2 suppresses divergent repeat-mediated deletions (RMDs). C_LIO_LIMLH1-PMS2 promotes polar resolution of sequence divergence. C_LIO_LISeveral mutants of MLH1-PMS2 affect both aspects of RMDs. C_LIO_LISome MLH1-PMS2 mutants affect only one aspect of RMDs. C_LIO_LISuppression of RMDs vs. polar resolution of divergence appear distinct. C_LI

molecular biology↗

bioRxiv2023

RAD52 and ERCC6L/PICH have a compensatory relationship for genome stability in mitosis.

The mammalian RAD52 protein is a DNA repair factor that has both strand annealing and recombination mediator activities, yet is dispensable for cell viability. To characterize genetic contexts that reveal dependence on RAD52 to sustain cell viability (i.e., synthetic lethal relationships), we performed genome-wide CRISPR knock-out screens. Subsequent secondary screening found that depletion of ERCC6L in RAD52-deficient cells causes reduced viability and elevated genome instability, measured as accumulation of 53BP1 into nuclear foci. Furthermore, loss of RAD52 causes elevated levels of anaphase ultrafine bridges marked by ERCC6L, and conversely depletion of ERCC6L causes elevated RAD52 foci both in prometaphase and interphase cells. These effects were enhanced with combination treatments using hydroxyurea and the topoisomerase II inhibitor ICRF-193, and the timing of these treatments are consistent with defects in addressing such stress in mitosis. Thus, loss of RAD52 appears to cause an increased reliance on ERCC6L in mitosis, and vice versa. Consistent with this notion, combined depletion of ERCC6L and disrupting G2/M progression via CDK1 inhibition causes a marked loss of viability in RAD52-deficient cells. We suggest that RAD52 and ERCC6L play compensatory roles in protecting genome stability in mitosis.

cell biology↗

bioRxiv2022

Mismatch resolution for repeat-mediated deletions show a polarity that is mediated by MLH1

Repeat-mediated deletions (RMDs) are a type of chromosomal rearrangement between two homologous sequences that causes loss of the sequence between the repeats, along with one of the repeats. Sequence divergence between repeats suppresses RMDs; the mechanisms of such suppression and of resolution of the mismatched bases remains poorly understood. We identified RMD regulators using a set of reporter assays in mouse cells that test two key parameters: repeat sequence divergence and the distances between one repeat and the initiating chromosomal double-strand break. We found that the mismatch repair factor MLH1 suppresses RMDs with sequence divergence in a manner that is epistatic with the mismatch repair factors MSH2 and MSH6, and which is dependent on residues in MLH1 and its binding partner PMS2 that are important for nuclease activity. Additionally, we found that resolution of mismatches in the RMD product have a specific polarity, where mismatched bases that are proximal to the chromosomal break end are preferentially removed. Moreover, we found that the MLH1 endonuclease domain is important for this polarity of mismatch resolution. Finally, we identified distinctions between MLH1 vs. TOP3 in regulation of RMDs. We suggest that MLH1 suppresses RMDs with sequence divergence, while also promoting directional mismatch resolution.

molecular biology↗

bioRxiv2021

The DNAPKcs long-range C-NHEJ complex is required for blunt DNA end joining when XLF is weakened

Canonical non-homologous end joining (C-NHEJ) factors can assemble into a long-range (LR) complex with DNA ends relatively far apart that contains DNAPKcs, XLF, XRCC4, LIG4, and the KU heterodimer and a short-range (SR) complex lacking DNAPKcs that has the ends positioned for ligation. Since the SR complex can form de novo, the role of the LR complex (i.e., DNAPKcs) for chromosomal EJ is unclear. We have examined EJ of chromosomal blunt DNA double-strand breaks (DSBs), and found that DNAPKcs is significantly less important than XLF and XRCC4 for such EJ. However, weakening XLF via disrupting interaction interfaces (e.g., disrupting the XLF homodimer interface) causes a marked requirement for DNAPKcs, its kinase activity, and its ABCDE-cluster autophosphorylation sites for blunt DSB EJ. In contrast, other aspects of genome maintenance are sensitive to DNAPKcs kinase inhibition in a manner that is not further enhanced by XLF loss (i.e., suppression of homology-directed repair and structural variants, and IR-resistance). We suggest that DNAPKcs is required to position a weakened XLF in an LR complex that can transition into a functional SR complex for blunt DSB EJ, but also has distinct functions for other aspects of genome maintenance.

molecular biology↗

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↗