Biology subjects

Sadeghi, C.

Publications and source records attributed to Sadeghi, C..

3 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↗