Biology subjects

Frock, R. L.

Publications and source records attributed to Frock, R. L..

6 recordsLinked to original sources

bioRxiv2024

ATM and 53BP1 regulate alternative end joining-mediated V(D)J recombination

G0/G1-phase alternative end joining (A-EJ) is a recently defined mutagenic pathway characterized by resected deletion and translocation joints that are predominantly direct and are distinguished from A-EJ in cycling cells which rely much more on microhomology-mediated end joining (MMEJ). Using chemical and genetic approaches, we systematically evaluate potential A-EJ factors and DNA damage response (DDR) genes to support this mechanism by mapping the repair fates of RAG1/2-initiated DSBs in the context of Ig{kappa} locus V-J recombination and chromosome translocation. Our findings highlight a polymerase theta-independent Parp1-XRCC1/Lig3 axis as central A-EJ components, supported by 53BP1 in the context of an ATM-activated DDR. Mechanistically, we demonstrate varied changes in short-range resection, MMEJ, and translocation, imposed by compromising specific DDR activities, which include polymerase alpha, ATR, DNA2, and Mre11. This study advances our understanding of DNA damage repair within the 53BP1 regulatory domain and the RAG1/2 post-cleavage complex.

molecular biology↗

bioRxiv2024

DNA-PKcs Suppresses Illegitimate Chromosome Rearrangements

Two DNA repair pathways, non-homologous end joining (NHEJ) and alternative end joining (A-EJ), are involved in V(D)J recombination and chromosome translocation. Previous studies reported distinct repair mechanisms for chromosome translocation, with NHEJ involved in human and A-EJ in mice predominantly. NHEJ depends on DNA-PKcs, a critical partner in synapsis formation and downstream component activation. While DNA-PKcs inhibition promotes chromosome translocations harboring microhomologies in mice, its synonymous effect in humans is not known. We find partial DNA-PKcs inhibition in human cells leads to increased translocations and the continued involvement of a dampened NHEJ. In contrast, complete DNA-PKcs inhibition substantially increased microhomology-mediated end joining (MMEJ), thus bridging the two different translocation mechanisms between human and mice. Similar to a previous study on Ku70 deletion, DNA-PKcs deletion in G1/G0-phase mouse progenitor B cell lines, significantly impairs V(D)J recombination and generated higher rates of translocations as a consequence of dysregulated coding and signal end joining. Genetic DNA-PKcs inhibition suppresses NHEJ involvement entirely, with repair phenotypically resembling Ku70-deficient A-EJ. In contrast, we find DNA-PKcs necessary in generating the near-excusive MMEJ associated with Lig4 deficiency. Our study underscores DNA-PKcs in suppressing illegitimate chromosome rearrangement while also contributing to MMEJ in both species.

genetics↗

bioRxiv2023

FLASH-RT does not affect chromosome translocations and junction structures beyond that of CONV-RT dose-rates

The molecular and cellular mechanisms driving the enhanced therapeutic ratio of ultra-high dose-rate radiotherapy (FLASH-RT) over slower conventional (CONV-RT) radiotherapy dose-rate remain to be elucidated. However, attenuated DNA damage and transient oxygen depletion are among several proposed models. Here, we tested whether FLASH-RT under physioxic (4% O2) and hypoxic conditions ([≤]2% O2) reduces genome-wide translocations relative to CONV-RT and whether any differences identified revert under normoxic (21% O2) conditions. We employed high-throughput rejoin and genome-wide translocation sequencing (HTGTS-JoinT-seq), using S. aureus and S. pyogenes Cas9 "bait" DNA double strand breaks (DSBs), to measure differences in bait-proximal repair and their genome-wide translocations to "prey" DSBs generated by electron beam CONV-RT (0.08-0.13Gy/s) and FLASH-RT (1x102-5x106 Gy/s), under varying ionizing radiation (IR) doses and oxygen tensions. Normoxic and physioxic irradiation of HEK293T cells increased translocations at the cost of decreasing bait-proximal repair but were indistinguishable between CONV-RT and FLASH-RT. Although no apparent increase in chromosome translocations was observed with hypoxia-induced apoptosis, the combined decrease in oxygen tension with IR dose-rate modulation did not reveal significant differences in the level of translocations nor in their junction structures. Thus, Irrespective of oxygen tension, FLASH-RT produces translocations and junction structures at levels and proportions that are indistinguishable from CONV-RT.

molecular biology↗

bioRxiv2022

Shifted PAMs generate DNA overhangs and enhance SpCas9 post-catalytic complex dissociation

Streptococcus pyogenes CRISPR-Cas9 (SpCas9) stabilizes each strand of a DNA bubble via distinct interactions, forming a stable ~20 bp R-loop on the complementary strand but relying on narrower protein-DNA interactions to bind the non-complementary strand with 5 NGG protospacer adjacent motif (PAM). The enzymes HNH endonuclease domain cleaves DNA within the R-loop, and its RuvC endonuclease cleaves 5 to the NGG and opposite the HNH cleavage site to generate blunt DNA ends. We show that this nucleoprotein complex can be mechanically strained by shifting the RNA:DNA hybrid and that the HNH cleavage site tracks this shift but the RuvC cleavage site does not, resulting in overhanging DNA ends. This is observed using cleavage in living cells and sequencing assays to characterize overhangs generated by stressed complexes, as well as single-molecule cleavage assays to characterize differential cleavage kinetics of HNH and RuvC endonucleases in stressed complexes as well as complex disassembly. Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the SpCas9 complex responds to internal mechanical strain by robustly generating overhanging, rather than blunt, DNA ends. Internal mechanical strain is generated by increasing or decreasing the spacing between the RNA:DNA hybrid and the downstream canonical PAM. Up to two-base 3 overhangs can be robustly generated via a two-base increase in the distance between hybrid and PAM. We also use single-molecule experiments to reconstruct the full course of the CRISPR-SpCas9 reaction in real-time, monitoring and quantifying the R-loop formation, the first and second DNA incision events, and the post-catalytic complex dissociation. Complex dissociation and release of broken DNA ends appears to be a rate-limiting step of the reaction, and strained SpCas9 are sufficiently destabilized so as to rapidly dissociate after formation of broken DNA ends.

genomics↗

bioRxiv2021

Ku70 suppresses alternative end-joining in G1-arrested progenitor B cells

Classical nonhomologous end-joining (C-NHEJ) repairs DNA double-stranded breaks (DSBs) throughout interphase but predominates in G1-phase when homologous recombination is unavailable. Complexes containing the Ku70/80 ("Ku") and XRCC4/Ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While XRCC4/Ligase IV are absolutely required for joining RAG1/2-endonucease ("RAG")-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Ligase IV also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Ligase IV-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAG-generated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally end-joining suppressed due to Ku-dependent blockage of A-EJ, potentially, in association with G1-phase down-regulation of Ligase1. Finally, we suggest that differential impacts of Ku-deficiency versus Lig4-deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-defcient developing lymphocyte and neuronal cells. Significance StatementAlternative end-joining (A-EJ) is implicated in oncogenic translocations and mediating DNA double-strand break (DSB) repair in cycling cells when classical nonhomologous endjoining (C-NHEJ) factors of the C-NHEJ Ligase complex are absent. However, V(D)J recombination-associated DSBs that occur in G1 cell cycle-phase progenitor lymphocytes are joined exclusively by the C-NHEJ pathway. Until now, however, the overall mechanisms that join general DSBs in G1-phase progenitor B cells had not been fully elucidated. Here, we report that Ku, a core C-NHEJ double-strand break recognition complex, directs repair of a variety of different targeted DSBs towards C-NHEJ and suppresses A-EJ in G1-phase cells. We suggest this Ku activity explains how Ku-deficiency can rescue the neuronal development and embryonic lethality phenotype of Ligase 4-deficient mice.

genetics↗

bioRxiv2018

Parp3 promotes long-range end-joining in murine cells

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classical- and alternative-nonhomologous end joining factors (NHEJ). We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. In contrast to c-NHEJ factors, we show here that Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class switch recombination in primary B cells and inversions in tail fibroblasts that generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting Parp1 may suppress DSB processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

cancer biology↗