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Human RECQL5beta stimulates flap endonuclease 1.

Speina E, Dawut L, Hedayati M, Wang Z, May A, Schwendener S, Janscak P, Croteau DL, Bohr VA - Nucleic Acids Res. (2010)

Bottom Line: Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage.Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes.This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.

View Article: PubMed Central - PubMed

Affiliation: National Institute on Aging, National Institutes of Health, 251 Bayview Blvd, Baltimore, MD 21224, USA.

ABSTRACT
Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5beta, with the human flap endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5beta dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes. This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.

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RECQL5β and FEN1 interact directly. Binding of FEN1 from 293T extracts (A) or purified FEN1 (B) to chitin beads coated with CBD or RECQL5β-CBD. (C) Diagram of GST-FEN1 fragments used to define which domain of FEN1 RECQL5β interacts with. (D) Binding of GST fused to various C-terminal fragments of FEN1 to chitin beads coated with RECQL5β-CBD.
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Figure 7: RECQL5β and FEN1 interact directly. Binding of FEN1 from 293T extracts (A) or purified FEN1 (B) to chitin beads coated with CBD or RECQL5β-CBD. (C) Diagram of GST-FEN1 fragments used to define which domain of FEN1 RECQL5β interacts with. (D) Binding of GST fused to various C-terminal fragments of FEN1 to chitin beads coated with RECQL5β-CBD.

Mentions: To determine whether RECQL5β and FEN1 interact physically, we performed affinity pull-down assays. RECQL5β was expressed in bacteria as a fusion with a CBD tag and bound to chitin beads. The beads were subsequently incubated with either total extract of exponentially growing human embryonic kidney cells HEK293T or with purified FEN1 protein. We found that endogenous FEN1 was bound to RECQL5β beads, but not to control beads coated with CBD (Figure 7A). Recombinant FEN1 was found to be bound to both RECQL5β beads and beads coated with CBD only. However, the affinity of FEN1 for the RECQL5β beads was higher than that for the control, indicating a specific interaction (Figure 7B).Figure 7.


Human RECQL5beta stimulates flap endonuclease 1.

Speina E, Dawut L, Hedayati M, Wang Z, May A, Schwendener S, Janscak P, Croteau DL, Bohr VA - Nucleic Acids Res. (2010)

RECQL5β and FEN1 interact directly. Binding of FEN1 from 293T extracts (A) or purified FEN1 (B) to chitin beads coated with CBD or RECQL5β-CBD. (C) Diagram of GST-FEN1 fragments used to define which domain of FEN1 RECQL5β interacts with. (D) Binding of GST fused to various C-terminal fragments of FEN1 to chitin beads coated with RECQL5β-CBD.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2875029&req=5

Figure 7: RECQL5β and FEN1 interact directly. Binding of FEN1 from 293T extracts (A) or purified FEN1 (B) to chitin beads coated with CBD or RECQL5β-CBD. (C) Diagram of GST-FEN1 fragments used to define which domain of FEN1 RECQL5β interacts with. (D) Binding of GST fused to various C-terminal fragments of FEN1 to chitin beads coated with RECQL5β-CBD.
Mentions: To determine whether RECQL5β and FEN1 interact physically, we performed affinity pull-down assays. RECQL5β was expressed in bacteria as a fusion with a CBD tag and bound to chitin beads. The beads were subsequently incubated with either total extract of exponentially growing human embryonic kidney cells HEK293T or with purified FEN1 protein. We found that endogenous FEN1 was bound to RECQL5β beads, but not to control beads coated with CBD (Figure 7A). Recombinant FEN1 was found to be bound to both RECQL5β beads and beads coated with CBD only. However, the affinity of FEN1 for the RECQL5β beads was higher than that for the control, indicating a specific interaction (Figure 7B).Figure 7.

Bottom Line: Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage.Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes.This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.

View Article: PubMed Central - PubMed

Affiliation: National Institute on Aging, National Institutes of Health, 251 Bayview Blvd, Baltimore, MD 21224, USA.

ABSTRACT
Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated with elevated cancer risk. RECQL1 and RECQL5 have not been linked to any human disorder yet; cells devoid of RECQL1 and RECQL5 display increased chromosomal instability. Here, we report the physical and functional interaction of the large isomer of RECQL5, RECQL5beta, with the human flap endonuclease 1, FEN1, which plays a critical role in DNA replication, recombination and repair. RECQL5beta dramatically stimulates the rate of FEN1 cleavage of flap DNA substrates. Moreover, we show that RECQL5beta and FEN1 interact physically and co-localize in the nucleus in response to DNA damage. Our findings, together with the previous literature on WRN, BLM and RECQL4's stimulation of FEN1, suggests that the ability of RecQ helicases to stimulate FEN1 may be a general feature of this class of enzymes. This could indicate a common role for the RecQ helicases in the processing of oxidative DNA damage.

Show MeSH
Related in: MedlinePlus