Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases
- University of Zurich, Switzerland
- University of Leipzig, Germany
Figures
Figure 1 with 2 supplements
Human DNA2 preferentially binds and degrades 5’ terminated ssDNA.
(A) A schematic representation of the recombinant hDNA2 protein used in this study. The polypeptide contains an N-terminal 6xHis- and a C-terminal FLAG affinity tag. The positions of the mutations …
Figure 1—figure supplement 1
Human RPA guides the hDNA2 nuclease to 5’ terminated ssDNA.
(A–C) Representative 10% polyacrylamide gels stained with Coomassie blue showing samples from (A) wild type hDNA2, (B) helicase-deficient hDNA2 K654R, and (C) nuclease- and helicase-deficient hDNA2 …
Figure 1—figure supplement 2
hDNA2 binds ssDNA.
(A–F) Representative 6% polyacrylamide gels showing the binding of nuclease-deficient hDNA2 D277A to various 32P-labeled oligonucleotide-based substrates. The blue triangle indicates the position of …
Figure 2
hDNA2 D277A shows DNA structure-dependent ATPase activity.
(A) Apparent ATP turnover number kcat with various DNA cofactors, including short 5’ overhang* (19 nt/31 bp), long 5’ overhang** (30 nt/31 bp), Y-structure (19 nt/ 31 bp), 3’ overhang (19 nt/ 31 …
Figure 3 with 1 supplement
hDNA2 D277A unwinds kilobase-lengths of dsDNA.
(A) Representative 1% agarose gel showing the helicase activity of hDNA2 D277A on λDNA/HindIII substrate with 346 nM hRPA. DNA unwinding leads to products that co-migrate with heat-denatured …
Figure 3—figure supplement 1
hDNA2 D277A unwinds plasmid- and oligonucleotide-based DNA substrates.
(A) Representative 1% agarose gel showing hDNA2 D277A helicase activity on a λDNA/HindIII substrate in a time-course experiment with 346 nM hRPA. Heat, heat-denatured DNA substrate. (B) …
Figure 4 with 1 supplement
Single molecule experiments reveal highly processive DNA unwinding by hDNA2 D277A.
(A) A sketch of the magnetic tweezers assay. (B) Representative DNA unwinding events (n = 7, colored) catalyzed by hDNA2 D277A at 22 ± 3 pN force. Experiments were conducted at 37°C in a reaction …
Figure 4—figure supplement 1
Single molecule experiments reveal that DNA unwinding by hDNA2 D277A is dependent on ATP and hRPA.
Experiments were carried out as in Figure 4B by adding hDNA2 D277A but omitting (A) both ATP and hRPA, (B) ATP or (C) hRPA only. While no activity was observed at all in the absence of ATP …
Figure 5 with 1 supplement
hDNA2 nuclease and helicase activities are regulated by ssDNA-binding proteins.
(A) Representative 1% agarose gels showing the helicase activity of hDNA2 D277A supplemented with indicated ssDNA-binding proteins on a 32P-labeled 2.7 kbp-long dsDNA substrate. (B) Quantitation of …
Figure 5—figure supplement 1
hDNA2 nuclease and helicase activities are regulated by ssDNA-binding proteins.
10% SDS-PAGE stained with Coomassie blue showing the purification procedures of (A) mtSSB and (B) the SOSS complex. PP, PreScission Protease. The ssDNA-binding properties of (C) mtSSB and (D) SOSS …
Figure 6 with 1 supplement
hDNA2 synergizes with WRN and BLM in the degradation of dsDNA.
Representative 1% agarose gels showing dsDNA degradation by wild type or helicase-deficient hDNA2 K654R variant with (A) WRN or (B) BLM. The reactions were supplemented with 50 mM NaCl and 215 nM …
Figure 6—figure supplement 1
Purification of WRN and BLM proteins.
(A) 10% SDS-PAGE stained with Coomassie blue showing the purification procedure of wild type WRN. PP, PreScission Protease. (B) 10% SDS-PAGE stained with Coomassie showing wild type WRN, …
Figure 7 with 1 supplement
The helicase activity of hDNA2 functionally integrates with BLM or WRN helicases.
(A) Quantitation of 2.7 kbp-long dsDNA unwinding by yDna2 E675A or Sgs1 with 400 nM of yeast RPA. Reactions were supplemented with 100 mM sodium acetate and 5 mM magnesium acetate and incubated at …
Figure 7—figure supplement 1
The functional integration of the helicase activity of hDNA2 is specific for WRN and BLM.
(A) Representative 1% agarose gel showing that the helicase-deficient mutants of various DNA helicases are not able to unwind a 2.7 kbp-long dsDNA substrate. (B) Representative 1% agarose gel …
Additional files
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Supplementary file 1
DNA sequences used in this study.
(A) Codon-optimized nucleotide sequence of hDNA2 gene for the expression in Sf9 cells. (B) Sequences of oligonucleotides used in this study. (C) Oligonucleotide-based DNA substrates used in this study.
- https://doi.org/10.7554/eLife.18574.016
