The intrinsically disordered amino-terminal region of human RecQL4: multiple DNA-binding domains confer annealing, strand exchange and G4 DNA binding

Abstract

Human RecQL4 belongs to the ubiquitous RecQ helicase family. Its N-terminal region represents the only homologue of the essential DNA replication initiation factor Sld2 of Saccharomyces cerevisiae, and also participates in the vertebrate initiation of DNA replication. Here, we utilized a random screen to identify N-terminal fragments of human RecQL4 that could be stably expressed in and purified from Escherichia coli. Biophysical characterization of these fragments revealed that the Sld2 homologous RecQL4 N-terminal domain carries large intrinsically disordered regions. The N-terminal fragments were sufficient for the strong annealing activity of RecQL4. Moreover, this activity appeared to be the basis for an ATP-independent strand exchange activity. Both activities relied on multiple DNA-binding sites with affinities to single-stranded, double-stranded and Y-structured DNA. Finally, we found a remarkable affinity of the N-terminus for guanine quadruplex (G4) DNA, exceeding the affinities for other DNA structures by at least 60-fold. Together, these findings suggest that the DNA interactions mediated by the N-terminal region of human RecQL4 represent a central function at the replication fork. The presented data may also provide a mechanistic explanation for the role of elements with a G4-forming propensity identified in the vicinity of vertebrate origins of DNA replication.

Saccharomyces cerevisiae Hrq1 helicase activity is affected by the sequence but not the length of single-stranded DNA

“…However, Hrq1 unwinding of forks with poly(dT)-tails of 25-, 50-, and 75-nt in length was indistinguishable, indicating that long (!70 nt) 3'-tails are not required for robust helicase activity. Finally, we demonstrated that the strong DNA strand annealing and exchange activities previously reported for RecQ4 [5] are absent in Hrq1.…”

“…Strand annealing activity is attributed to the Sld2-like N-terminal domain of RecQ4 [5,17], which is further supported by the demonstrated DNA annealing activity of S. cerevisiae Sld2 [4]. We previously found no hint of DNA strand annealing by Hrq1 [8], presumably because it does not contain a Sld2-like domain.…”

“…RecQ4 helicase assays were performed in the presence of 15 nM cold ssDNA trap [14]. ATP was required to observe unwinding, suggesting that strand exchange was not the activity being measured (data not shown) [5]. Reactions were incubated at 37 C for 30 min and stopped with the addition of 1Â Stop-Load dye (5% glycerol, 20 mM EDTA, 0.05% SDS, and 0.25% bromophenol blue) supplemented with 400 mg/mL SDS-Proteinase K followed by a 1-min incubation at 37 C. Unwound DNA was separated on 8% 19:1 acrylamide:bis-acrylamide gels in TBE buffer at 10 V/cm and imaged as for the DNA binding assay.…”

“…These assays were performed in the same conditions as for annealing activity, and products were separated on 12% 19:1 acrylamide:bisacrylamide gels in TBE buffer at 10 V/cm and imaged as above. The fork oligonucleotides used in the strand annealing and strand exchange assays were identical to those used previously to identify RecQ4-mediated strand exchange activity [5].…”

“…For instance, the presence of a Sld2-like domain (an essential replication initiation factor in yeast) at the N-terminus of RecQ4 makes mutational analysis difficult as mutations could disrupt DNA replication [2], which is independent of RecQ4-mediated DNA repair [3]. The Sld2-like domain also confounds biochemical analysis because Saccharomyces cerevisiae Sld2 is a known annealase [4], and the Sld2-like portion of RecQ4 plays a significant role in DNA annealing activity [5]. This ability of RecQ4 to anneal complementary DNA substrates is so robust that it initially masked the DNA unwinding activity of the enzyme [6], making RecQ4 helicase activity difficult to analyze.…”