Nucleic Acids Research最新文献

Type V-F CRISPR-Cas12f is a group of hypercompact RNA-guided nucleases that present a versatile in vivo delivery platform for gene therapy. Upon target recognition, Acidibacillus sulfuroxidans Cas12f (AsCas12f1) distinctively engenders three DNA break sites, two of which are located outside the protospacer. Combining ensemble and single-molecule approaches, we elucidate the molecular details underlying AsCas12f1-mediated DNA cleavages. We find that following the protospacer DNA unwinding and non-target strand (NTS) DNA nicking, AsCas12f1 surprisingly carries out bidirectional exonucleolytic cleavage from the nick. Subsequently, DNA unwinding is extended to the out-of-protospacer region, and AsCas12f1 gradually digests the unwound DNA beyond the protospacer. Eventually, the single endonucleolytic target-strand DNA cleavage at 3 nt downstream of the protospacer readily dissociates the ternary AsCas12f1-sgRNA-DNA complex from the protospacer adjacent motif-distal end, leaving a staggered double-strand DNA break. The coupling between the unwinding and cleavage of both protospacer and out-of-protospacer DNA is promoted by Mg2+. Kinetic analysis on the engineered AsCas12f1-v5.1 variant identifies the only accelerated step of the protospacer NTS DNA trimming within the sequential DNA cleavage. Our findings provide a dynamic view of AsCas12f1 catalyzing DNA unwinding-coupled nucleolytic cleavage and help with practical improvements of Cas12f-based genome editing tools.

To ensure the integrity of our genetic code, a coordinated network of signalling and repair proteins, known as the DNA damage response (DDR), detects and repairs DNA insults, the most toxic being double-strand breaks (DSBs). Tudor interacting repair regulator (TIRR) is a key factor in DSB repair, acting through its interaction with p53 binding protein 1 (53BP1). TIRR is also an RNA binding protein, yet its role in RNA regulation during the DDR remains elusive. Here, we show that TIRR selectively binds to a subset of messenger RNAs (mRNAs) in response to DNA damage. Upon DNA damage, TIRR interacts with the nuclear export protein Exportin-1 through a nuclear export signal. Furthermore, TIRR plays a crucial role in the modulation of RNA processing bodies (PBs). TIRR itself and TIRR-bound RNA co-localize with PBs, and TIRR depletion results in nuclear RNA retention and impaired PB formation. We also suggest a potential link between TIRR-regulated RNA export and efficient DDR. This work reveals intricate involvement of TIRR in orchestrating mRNA nuclear export and storage within PBs, emphasizing its significance in the regulation of RNA-mediated DDR.

Mechanisms underlying p53-mediated protection of the replicating genome remain elusive, despite the quintessential role of p53 in maintaining genomic stability. Here, we uncover an unexpected function of p53 in curbing replication stress by limiting PARP1 activity and preventing the unscheduled degradation of deprotected stalled forks. We searched for p53-dependent factors and elucidated RRM2B as a prime factor. Deficiency in p53/RRM2B results in the activation of an NRF2 antioxidant transcriptional program, with a concomitant elevation in basal PARylation in cells. Dissecting the consequences of p53/RRM2B loss revealed a crosstalk between redox metabolism and genome integrity that is negotiated through a hitherto undescribed NRF2-PARP1 axis, and pinpoint G6PD as a primary oxidative stress-induced NRF2 target and activator of basal PARylation. This study elucidates how loss of p53 could be destabilizing for the replicating genome and, importantly, describes an unanticipated crosstalk between redox metabolism, PARP1 and p53 tumor suppressor pathway that is broadly relevant in cancers and can be leveraged therapeutically.

In many bacteria, the essential factors Rho and NusG mediate termination of synthesis of nascent transcripts (including antisense RNAs) that are not being simultaneously translated. It has been proposed that in Rho's absence toxic RNA-DNA hybrids (R-loops) may be generated from nascent untranslated transcripts, and genome-wide mapping studies in Escherichia coli have identified putative loci of R-loop formation from more than 100 endogenous antisense transcripts that are synthesized only in a Rho-deficient strain. Here we provide evidence that engineered expression in wild-type E. coli of several such individual antisense regions on a plasmid or the chromosome generates R-loops that, in an RNase H-modulated manner, serve to disrupt genome integrity. Rho inhibition was associated with increased prevalence of antisense R-loops also in Xanthomonas oryzae pv. oryzae and Caulobacter crescentus. Our results confirm the essential role of Rho in several bacterial genera for prevention of toxic R-loops from pervasive yet cryptic endogenous antisense transcripts. Engineered antisense R-looped regions may be useful for studies on both site-specific impediments to bacterial chromosomal replication and the mechanisms of their resolution.