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. 2022:673:169-190.
doi: 10.1016/bs.mie.2022.03.031. Epub 2022 Apr 9.

Bulk phase biochemistry of PIF1 and RecQ4 family helicases

Prasangi Rajapaksha  1 , Robert H Simmons 3rd  1 , Spencer J Gray  1 , David J Sun  1 , Phoebe Nguyen  1 , David G Nickens  1 , Matthew L Bochman  2
Affiliations

Bulk phase biochemistry of PIF1 and RecQ4 family helicases

Prasangi Rajapaksha et al. Methods Enzymol. 2022.

Abstract

DNA helicases are involved in nearly all facets of genome integrity, and in humans, mutations in helicase-encoding genes are often linked to diseases of genomic instability. Two highly studied and evolutionarily conserved helicase families are the PIF1 and RecQ helicases. Enzymes in these families have known roles in DNA replication, recombination, and repair, as well as telomere maintenance, DNA recombination, and transcription. Although genetics, structural biology, and a variety of other techniques have been used to study these helicases, ensemble analyses of their basic biochemical activities such as DNA binding, ATP hydrolysis, and DNA unwinding have made significant contributions to our understanding of their physiological roles. Here, we present general methods to generate recombinant proteins from both helicase families, as well as standard biochemical assays to investigate their activities on DNA.

Keywords: ATPase; DNA binding; Helicase; Hrq1; PIF1; RecQ.

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Figures

Fig. 1
Fig. 1
NADH-coupled ATPase assay. In this assay, a helicase (+/− DNA, not shown) binds and hydrolyzes ATP into ADP. To maintain the ATP at a constant concentration, phosphoenol pyruvate (PEP) and pyruvate kinase (PK) are used as an ATP regeneration system, recycling ADP into ATP and generating pyruvate. This reaction is coupled to lactate production by the action of lactate dehydrogenase (LDH) on pyruvate in the presence of NADH. The conversion of NADH to NAD+ is monitored by recording the decrease in absorbance at 340 nm (A340). One ATP is hydrolyzed for every NADH oxidized.
Fig. 2
Fig. 2
Example helicase, DNA annealing, and stand exchange assays. (A) Conversion of the fork substrate into ssDNA by unwinding the duplex portion of the fork substrate is catalyzed by the addition of a DNA helicase in a concentration-dependent manner (1–100 nM). The boiled control (+) shows the position at which the ssDNA should migrate through the gel, and the unboiled control (−) shows where the fork substrate migrates (t = 30 min endpoints for each control). The fork substrate shown was created by annealing oligonucleotides MB1057 and MB1058 (Table 1). (B) Annealing of two partially complementary ssDNAs (MB1057 and MB1058) into a DNA fork by the RECQL4 (100 nM; RecQ4) helicase. Here, the annealed product (i.e., the fork) was boiled (+) to indicate the position where the unannealed ssDNAs migrate through the gel, and the unboiled (−) control indicates the migration position of the annealed fork. (C) Strand exchange catalyzed by 50 nM RECQL4. In this assay, the unlabeled partially complementary oligonucleotide (blue, MB1058) that forms half of the fork substrate in (A) and (B) can be exchanged for a completely complementary unlabeled oligonucleotide (black, MB1059) to produce blunt dsDNA, which migrates more quickly in the gel than the fork. The images in (B) and (C) are adapted from Rogers, C. M., & Bochman, M. L. (2017). Saccharomyces cerevisiae Hrq1 helicase activity is affected by the sequence but not the length of single-stranded DNA. Biochemical and Biophysical Research Communications, 486(4), 1116–1121. doi:10.1016/j.bbrc.2017.04.003.
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