Helicase HELQ: Molecular Characters Fit for DSB Repair Function.

The protein sequence and spatial structure of DNA helicase HELQ are highly conserved, spanning from archaea to humans. Aside from its helicase activity, which is based on DNA binding and translocation, it has also been recently reconfirmed that human HELQ possesses DNA-strand-annealing activity, similar to that of the archaeal HELQ homolog StoHjm. These biochemical functions play an important role in regulating various double-strand break (DSB) repair pathways, as well as multiple steps in different DSB repair processes. HELQ primarily facilitates repair in end-resection-dependent DSB repair pathways, such as homologous recombination (HR), single-strand annealing (SSA), microhomology-mediated end joining (MMEJ), as well as the sub-pathways' synthesis-dependent strand annealing (SDSA) and break-induced replication (BIR) within HR. The biochemical functions of HELQ are significant in end resection and its downstream pathways, such as strand invasion, DNA synthesis, and gene conversion. Different biochemical activities are required to support DSB repair at various stages. This review focuses on the functional studies of the biochemical roles of HELQ during different stages of diverse DSB repair pathways.

Human HELQ regulates DNA end resection at DNA double-strand breaks and stalled replication forks.

Following a DNA double strand break (DSB), several nucleases and helicases coordinate to generate single-stranded DNA (ssDNA) with 3' free ends, facilitating precise DNA repair by homologous recombination (HR). The same nucleases can act on stalled replication forks, promoting nascent DNA degradation and fork instability. Interestingly, some HR factors, such as CtIP and BRCA1, have opposite regulatory effects on the two processes, promoting end resection at DSB but inhibiting the degradation of nascent DNA on stalled forks. However, the reason why nuclease actions are regulated by different mechanisms in two DNA metabolism is poorly understood. We show that human HELQ acts as a DNA end resection regulator, with opposing activities on DNA end resection at DSBs and on stalled forks as seen for other regulators. Mechanistically, HELQ helicase activity is required for EXO1-mediated DSB end resection, while ssDNA-binding capacity of HELQ is required for its recruitment to stalled forks, facilitating fork protection and preventing chromosome aberrations caused by replication stress. Here, HELQ synergizes with CtIP but not BRCA1 or BRCA2 to protect stalled forks. These findings reveal an unanticipated role of HELQ in regulating DNA end resection at DSB and stalled forks, which is important for maintaining genome stability.

The RNA helicase Ski2 in the fungal pathogen Cryptococcus neoformans highlights key roles in azoles resistance and stress tolerance.

The yeast SKI (superkiller) complex was originally identified from cells that were infected by the M 'killer' virus. Ski2, as the core of the SKI complex, is a cytoplasmic cofactor and regulator of RNA-degrading exosome. The putative RNA helicase Ski2 was highly conserved from yeast to animals and has been demonstrated to play a key role in the regulation of RNA surveillance, temperature sensitivity, and growth in several yeasts but not yet in Cryptococcus neoformans (C. neoformans). Here, we report the identification of a gene encoding an equivalent Ski2 protein, named SKI2, in the fungal pathogen C. neoformans. To obtain insights into the function of Ski2, we created a mutant strain, ski2Δ, with the CRISPR-Cas9 editing tool. Disruption of SKI2 impaired cell wall integrity. Further investigations revealed the defects of the ski2Δ mutant in resistance to osmotic stresses and extreme growth temperatures. However, significantly, the ability to undergo invasive growth under nutrient-depleted conditions was increased in the ski2Δ mutant. More importantly, our results showed that the ski2Δ mutant exhibited slightly lower virulence and severe susceptibility to anti-ribosomal drugs by comparison to the wild type, but it developed multidrug resistance to azoles and flucytosine. By constructing the double deletion strain ski2Δafr1Δ, we verified that increased Afr1 in ski2Δ contributed to the azole resistance, which might be influenced by nonclassical small interfering RNA. Our work suggests that Ski2 plays critical roles in drug resistance and regulation of gene transcription in the yeast pathogen C. neoformans.