The HelQ human DNA repair helicase utilizes a PWI-like domain for DNA loading through interaction with RPA, triggering DNA unwinding by the HelQ helicase core

Abstract

Genome instability is a characteristic enabling factor for carcinogenesis. HelQ helicase is a component of human DNA maintenance systems that prevent or reverse genome instability arising during DNA replication. Here, we provide details of the molecular mechanisms that underpin HelQ function—its recruitment onto ssDNA through interaction with replication protein A (RPA), and subsequent translocation of HelQ along ssDNA. We describe for the first time a functional role for the non-catalytic N-terminal region of HelQ, by identifying and characterizing its PWI-like domain. We present evidence that this domain of HelQ mediates interaction with RPA that orchestrates loading of the helicase domains onto ssDNA. Once HelQ is loaded onto the ssDNA, ATP-Mg2+ binding in the catalytic site activates the helicase core and triggers translocation along ssDNA as a dimer. Furthermore, we identify HelQ-ssDNA interactions that are critical for the translocation mechanism. Our data are novel and detailed insights into the mechanisms of HelQ function relevant for understanding how human cells avoid genome instability provoking cancers, and also how cells can gain resistance to treatments that rely on DNA crosslinking agents.

HELQ is a dual-function DSB repair enzyme modulated by RPA and RAD51

“…Roopesh Anand 1,6 , Erika Buechelmaier 2,3,6 , Ondrej Belan 1 , Matthew Newton 1 , Aleksandra Vancevska 1 , Artur Kaczmarczyk 4,5 , Tohru Takaki 1 , David S. Rueda 4,5 ✉ , Simon N. Powell 2 ✉ & Simon J. Boulton 1 ✉ DNA double-stranded breaks (DSBs) are deleterious lesions, and their incorrect repair can drive cancer development 1 . HELQ is a superfamily 2 helicase with 3′ to 5′ polarity, and its disruption in mice confers germ cells loss, infertility and increased predisposition to ovarian and pituitary tumours [2][3][4] .…”

“…At the cellular level, defects in HELQ result in hypersensitivity to cisplatin and mitomycin C, and persistence of RAD51 foci after DNA damage 3 , 5 . Notably, HELQ binds to RPA and the RAD51-paralogue BCDX2 complex, but the relevance of these interactions and how HELQ functions in DSB repair remains unclear 3 , 5 , 6 . Here we show that HELQ helicase activity and a previously unappreciated DNA strand annealing function are differentially regulated by RPA and RAD51.…”

Helicase Q promotes homology-driven DNA double-strand break repair and prevents tandem duplications

“…The mechanism by which HELQ makes ssDNA available for base-pairing interactions is currently unknown, but primarily because of recent in vitro work we favour a function in removing potentially annealing-interfering ssDNA binding proteins such as RPA or RAD51. HELQ, purified from an archaeal or human source, was found to interact with RPA and able to modulate the RPA-DNA binding including its displacement from ssDNA 57 , 58 ; C. elegans HELQ-1 was shown to promote the disassembly of RAD51 from DNA in vitro 47 . Also, the helicase domain in POLQ, which is highly similar to HELQ, was capable of removing RPA from resected DSB ends in vitro to allow their annealing 37 .…”

Polymerase θ Coordinates Multiple Intrinsic Enzymatic Activities during DNA Repair

“…Purified ATPase proficient HELQ protein unwinds model replication fork substrates in vitro [ 49 ] without any of the issues surrounding the low specific activity of the pol θ N-terminal ATPase construct. Additional experimentation will be necessary to clarify why the pol θ ATPase lacks the strong enzymatic activity of HELQ [ 50 ], or the archaeal homolog HEL308, which can break the biotin–streptavidin interaction due to tenacious helicase and protein-stripping functions [ 51 ].…”

“…Dimerization is often observed for DNA repair enzymes able to synapse DSBs, such as DNA-PK or LIG4, but functional tetramerization aligns more with helicases catalyzing branch migration of 4-way DNA junctions [ 52 ]. Like the pol θ ATPase, HELQ was also shown to exist in solution as a tetramer by SEC-MALS, but emerging evidence implies that active complexes of the HELQ could exist as dimers, hinting that the tetrameric form might serve a regulatory purpose [ 50 ]. Identifying the functional biological assembly assumed by the pol θ ATPase domain in cells warrants further analysis.…”