Browsing by Subject "R-loop"

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Characterization of biological functions of human RNA-binding proteins in Poly(ADP-ribose) polymerase-1-regulated pathways
(2022-07-29) Lin, Wen-Ling (Ph. D. in biochemistry); Liu, Hung-wen, 1952-; Russell, Rick; Lee, Seongmin; Paull, Tanya T.; Fast, Walter L.
Poly(ADP-ribosy)lation (PARylation) can served as a scaffold for noncovalent interactions with various RNA binding proteins including helicases and ribosomal proteins, but many important questions remains regarding the molecular functions and biological roles through the interaction. Based on live-cell imaging assay, I found that DEAD-box helicase 18 (DDX18) and ribosomal protein S19 (RPS19) accumulated to DNA damage sites via PARP-1 activation, indicating their participation in PARP-1-dependent DNA damage repair. My results show that PARP-1 can mediate the association of DDX18 with R-loops thereby modulating R-loop homeostasis and R-loop-dependent DNA damage. DDX18 knockdown renders cells more sensitive to DNA damaging reagents. Furthermore, knockdown of DDX18 reduces R-loop-induced RPA32 and RAD51 foci formation in response to irradiation, and DDX18 depletion also leads to R-loop-induced γH2AX accumulation and genome instability. In addition, DDX18 knockdown abolishes DNA replication due to R-loop accumulation. Taken together, the data uncover new functions of PARP-1 mediated DDX18 in R-loop-mediated events. Here, I also identify RPS19 as a new regulator in DNA double-strand break (DSB) repair. RPS19 was recruited to the DNA damage regions through its putative PAR-binding motif, and the accumulation of RPS19 was PARP-1 dependent. In the absence of RPS19, cells showed reduced RAD51 foci formation, whereas γH2AX and RPA32 foci were increased. Furthermore, the accumulation of RPS19 at DNA damage sites were abolished during the treatment of ATR inhibitors but not ATM inhibitors. Upon X-ray irradiation, RPS19-knockdown cells also decreased phosphorylation of Chk1 at Ser345, which is the downstream of ATR. The results suggest the novel role of RPS19 in ATR pathway during DNA damage repair. In summary, these observations provide a further mechanistic understanding of RNA-binding proteins in PARP-1 mediated DNA damage responses.
Kinetic characterization of CRISPR-Cas12a DNA targeting
(2021-05-06) Strohkendl, Isabel Suzanna; Russell, Rick, 1969-; Finkelstein, Ilya J; Johnson, Arlen W; Johnson, Kenneth A; Taylor, David W
Cas12a is a type V class 2 CRISPR-Cas nuclease that has recently been repurposed as a precision DNA-cutting tool, alongside the type II Cas9. Their potential for biotechnological and genome editing applications has driven enthusiastic efforts to biochemically characterize their assembly and RNA-guided DNA targeting mechanisms. Two important obstacles that must be resolved before these nucleases can be used widespread are their specificity and efficiency; CRISPR nucleases are known to cut sequences that resemble their intended target, and within the cell—where our genomic DNA is packaged into protein-rich chromatin—it is unclear how target preferences are influenced. Here, we quantitatively study Acidaminococcus sp. Cas12a DNA targeting behavior in vitro using a kinetics-based approach. We begin by characterizing Cas12a binding and cleavage on matched and mismatch targets (Chapter 2), uncovering how Cas12a achieves such high levels of specificity despite cleaving DNA in a rate-limiting binding regime. We then characterize Cas12a DNA targeting on substrates that begin resemble genomic chromatin (Chapter 3). Lastly, we review the fundamentals of CRISPR nuclease specificity and how engineering efforts to develop a ‘high-fidelity’ nuclease should be focused (Chapter 4).